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Franchise News Search News Deregulation taskforce a 'game-changer' for small business
By Nick Hall | 16 Sep 2019 View comments
Prospective small business owners may be in for significant boost, after the Federal Government launched a new deregulation taskforce.
Announced on Thursday, the taskforce will work alongside state and territory governments, and businesses themselves to identify and address the most significant barriers to investment.
Ben Morton, Assistant Minister to the Prime Minister and Cabinet said the latest development was intended to help new business owners receive access to finance.
"This continues the Coalition Government's commitment to reduce red tape and unnecessary regulation, making it easier for businesses to invest, create jobs and grow the economy," Morton said in a statement.
"A better regulatory environment will help businesses lower their costs, save time and improve their competitiveness, while major infrastructure projects will help get us home sooner and safer."
Deregulation taskforce key points
According to the government, the deregulation taskforce will focus on three key areas of implementation. These include; the degree of regulatory complexity, the length of time for approvals and duplication across levels of government.
The announcement of the deregulation taskforce comes at an interesting time for the franchising sector.
A difficult period of lending, coupled with strong regulatory conditions have weakened the rate of franchise growth across a number of industries.
Speaking at the Franchise Council of Australia's recruitment forum in July, Hire a Hubby CEO Brendan Green said it was often a frustrating situation to have qualified, capable applicants who are unable continue in the process due to lending constraints.
"One of the biggest things is having finance solutions in the background," Green said. "If you've got someone who is a willing candidate, they might have the capacity but not the sophistication to acquire that money. It can be a real challenge.
Green suggested that as a result, a number of franchise brands had to turned to alternative lending or outside initiatives such as joint ventures to open the opportunity.
"If you take a look at the property market, which is where a lot of older Australians have made the money to get into business, younger people can't do that. So, we have to get them in in different ways, and we've found that initiatives like the joint venture process to be highly successful.
Small business 'crying out' for support
The government's new deregulation taskforce may ease conditions for a number of prospective entrepreneurs, however.
Kate Carnell, Australian Small Business and Family Enterprise Ombudsman (ASBFEO) suggested the new initiative would help to cut red tape, opening the door for future growth.
"Small businesses and family enterprises are crying out for less red tape and unnecessary regulation, so they can get on with the job of growing their business," Carnell said.
"The government's efforts to bring down the barriers to business investment and boost productivity are commendable. While we support the intentions of the deregulation taskforce in principle, understanding the detail of these proposals to ensure there are no unintended adverse consequences, is vital."
Despite throwing her support behind the new deregulation taskforce, Carnell warned that consultation between all parties was critical to the implementation's success.
"It's also critical the government consults widely as part of the deregulation process, which my office is looking forward to playing an active role in, to ensure the government engages fully with small business," she said.
"This has the potential to be a game-changer for Australia's small businesses and family enterprises."
access to finance deregulation taskforce federal government retail leasing small business starting a franchise
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Above $500,000


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Patent Troll has accused Apple of violating patents in FaceTime
Literally in February we wrote about the fact that Apple has to pay a fine of $625 million for patent infringement of the company VirnetX. At the time we talked on FaceTime and iMessage is, where was used the technology described in the disputed patent. Brand Apple services appear in a new lawsuit against the company from Cupertino. This time, the claimant is a Straight Path Group, alleging that FaceTime violates their copyrights.
On the iPhone and iPad maker faces the threat of new penalties: the American computer electronics giant is accused of violating five patents owned by Straight Path Group. The plaintiffs argue that FaceTime uses the ideas, many of which 20 years ago were implemented in them belonging to product WebPhone.
Given the patents system is affected by the push notification and the Protocol of establishing a session used in the messenger from Apple. In addition, the plaintiffs believe that the principle of hardware integration FaceTime with iPhone and iPad, was also borrowed from WebPhone.
Observers note that the nature of the activities Straight Path Group allows you to classify this company as a professional "patent trolls" — that is, those who specializiruetsya on filing patent lawsuits to large corporations.
Interestingly, in may other company VoIP-Pal has accused Apple of violating patents in FaceTime and iMessage. Just in case are two patents. They refer to "innovative" classification of attributes of the caller and routing system messages. The patents involved in the case, was acquired by VoIP-Pal operator Digifonica in 2013. Also, VoIP-Pal has filed a lawsuit against Verizon and AT&T. the Total amount of claims is $7 billion.
Tags: appleiPadiPhonePatents
PlayStation 5 will be the most powerful console in history Sony
What will be the new iPad Pro
How to trace the sending przekazania iPhone X
Next story Apple took part in a gay pride parade in San Francisco, employees donated a "rainbow" straps for Apple Watch
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redpajama_set_name: RedPajamaCommonCrawl


Actors like Jesse Williams continue to restore my belief in some of our black entertainers. Williams is the type of entertainer that is cut from the same cloth that brought us the likes of legendary figures such as Ozzie Davis, Ruby Dee, and Harry Belafonte. He's the type of black entertainer who is not afraid to speak truth to power. He is the type that sees an injustice and is not afraid to speak on the injustice unlike most of our other entertainers who have remained remarkably silent except for an occasional social media post.
During an appearance on CNN's State of the Union, Williams took the mainstream media to task for it's choice of narratives when it came to the murder of Mike Brown.
"We also have to talk about the narrative and making sure that we're starting at the beginning. You'll find that the people doing the oppressing always want to start the narrative at a convenient part, or always want to start the story in the middle. This started with a kid getting shot and killed and left in the street for four hours. I've never seen a white body left in the street for four hours in the sweltering heat. The cop doesn't call in the shooting, the body isn't put in an ambulance, it's shuttled away in some shady unmarked SUV.
This idea that because he stole a handful of cheap cigars, what's that $5? I've lived in white suburbs of this country for a long time, I know plenty of white kids who steal stuff from a convenience store. [There's] this idea that every time a black person does something, they automatically become a thug worthy of death when we don't own drug crimes. We're not the only ones who sell and do drugs all the time. We're not the only ones that steal and talk crazy to cops.


BRIGHT and BEAUTIFUL 2 bed, 2 bath corner suite in the heart of Richmond. Open floorplan with stainless steel appliances, granite countertops and beautifully added: crown mouldings, laminate throughout, closet organizers, interior doors and balcony screen door. Oceanwalk is centrally located with easy access to Vancouver & Canada Line. Short walking distance to: Olympic Oval, Steve Nash Gym, T & T Supermarket and more. Well maintained strata with amenities featuring a 9 hole putting green, rooftop garden, playground. gym, lounge, guest suites and live-in caretaker. Open house: Saturday, May 27 & Sunday May 28 from 2pm-4pm.


VirnetX is looking to squeeze a little more juice from Apple, pressing the company with another round of patent infringement lawsuits.
In fact, earlier this week a Texas court ordered Apple to pay the internet security software and technology company $368 million (UK£231, AUD$353) over patent violations involving FaceTime.
"We are extremely pleased with the outcome of our suit with Apple," VirnetX CEO Kendall Larsen said in a press release. "This victory further establishes the importance of our patent portfolio."
With this new round of infringement complaints coming, VirnetX is further testing the portfolio's worth.
The grievances are the same as its last suit, but now VirnetX is targeting the iPhone 5, iPad Mini, iPad 4, iPod Touch 5 and the latest desktop Macs. The company is looking to clean up the devices that were too new to be included in the first round.
VirnetX sought $708 million (UK£442, AUD$680) in damages, but had to settle for about half that amount after the court's ruling. The complaint included four networking patents filed between 2002 and 2011 that FaceTime allegedly infringed on.
Some of the patents cover: technology that creates a virtual private network between two computers, a protocol for securing communications using secure domain names, and a method that establishes a secure communications connection based on a domain name service request.
VirnetX is known for aggressively protecting its patents, going after big tech companies that infringe on its properties. In 2010, it won a $200 million (UK£125, AUD$192) claim against Microsoft.
VirnetX is also currently slugging it out with Cisco, Avaya and Siemens in infringement courts.
The multimillion dollar settlement shouldn't put too big a dent in Apple's billions in quarterly profits, though the suits could have other ramifications.
VirnetX's legal victories could back up claims filed with the U.S. International Trade Commission, leading to possible sales bans on some Apple products.
For now VirnetX is looking to take a victory lap in courts and skin a little bit more green off Apple.


This cabin has a sliding glass door out to deck overlooking Isle Bay where there's a picnic table with umbrella. The Kitchen is fully equipped with a stove, refrigerator, microwave, basic cooking and eating utensils, toaster and Mr. Coffee coffee pot. The bedroom has a queen size bed and a roll-a-way bed. The living area has a hide-a-bed futon couch for added sleeping. There's a flat screen TV w/Cable, fan and air conditioning.
RATES: $99/night – $594/week (for 2 people) Rates are based on number of people regardless of age.
$25 additional charge for 1 night stays (last minute reservations only).


The joy of omni-channel marketing is that it gives us many opportunities to connect with customers all along their journeys with our brands. Too often, though, we focus on the channels themselves and not on the strategies that drive the way we use those channels to achieve our marketing and business objectives.
Even with technology that does almost all the thinking for us, marketing remains an art and a science. It focuses first on goals and strategies, then puts them to use through coordinated tactical decisions.
If your marketing program uses several channels—digital channels like email, SMS, rich push, in-app messaging, web push, direct mail and social media—and you're not seeing the results you want or expect, use the three steps outlined below to refocus and recalibrate your efforts.
Top-down: Create goals, objectives and strategy to guide your decision-making efforts, including decisions on which channels to use and the tactics and technologies you need to put your strategy into effect, achieve your objectives and reach your goals.
Bottom-up: The tactics or technologies run the show. The results are usually a disconnected experience for the customer and a haphazard approach to meeting your business goals that wastes time and money.
Strategy is your guide to the omni-channel ecosystem. It helps you make informed decisions all along the way, from setting up and optimizing a coordinated messaging plan among channels to selecting the KPIs and testing strategies you need to measure and evaluate your results.
Without it, you flounder. In today's environment of paper-thin margins and budget scrutiny, no one can afford to waste time, money and resources.
It's easy to understand why so many marketers let technology take the lead. Marketing today is so complex that we yearn for a silver bullet—the one tool guaranteed to work every time.
Sad to say—there is no silver bullet. But having a well-thought-out strategy that supports your business goals and objectives will lead you to find the right tools every time.
What's the trouble with technology? Nothing. The growth marketing technology available to today's brands has amazing capabilities. But that's where the trouble starts.
Technology is tactical. You can achieve one-off success with it. But business thrives on consistent success. What drives this consistent success is having an objective, a strategy to achieve that objective and tactics and technology to carry out that strategy.
Technology needs guidance to drive the greatest results. That's why you need a workable strategy to make that technology help you achieve your goals and objectives.
The seamless, unified experience across channels is the dream and promise of omni-channel marketing, but it doesn't happen without data.
This includes the customer data you hold internally (demographic, behavioral and event) and data you acquire externally from third parties. This data demonstrates to your customers that you know them by serving personalized messages, whether in an email, a push notification, a remarketing ad or whatever the occasion calls for.
Ideally, this data is available to your systems in real time, using webhooks and other integrations rather than relying on batched data. All your databases must be connected so they can speak to each other—email, e-commerce, CRM, etc.
The email address becomes a central point of entry because you can use it to identify individual customers across channels using all the data you hold on them. That emphasizes the importance of having a visible, friction-free email opt-in process that stresses the benefits of joining your email program (and, subsequently, delivers on those benefits).
A successful omni-channel strategy assumes that no single channel is complete by itself. Email, for example, is a great channel to drive traffic toward the point of conversion, but it rarely succeeds in a vacuum.
A workable omni-channel strategy envisions all the points along the journey where your customer contacts your brand and responds in the appropriate channel. The chart below shows many points on a model customer journey—you can see right away that one channel is not the right source for every contact.
When your definition of success is a conversion—whether a purchase, a new customer account, a registration, a download, or a request—email works with other marketing channels to be optimized and converting well to succeed.
You can apply this to your website copy and organization, to search keywords, PPC campaigns and related landing pages, and in ads and banners you run on third-party sites in remarketing or network campaigns.
3. A holistic approach that incorporates all your marketing channels to collect data points along the customer journey and trigger messages in the appropriate channels.
This approach to omni-channel marketing takes time and effort. But, the payoff is a sustainable and reliable process that delivers success after success.


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redpajama_set_name: RedPajamaGithub


I'm new to your community and I'm overwhelmed how present Doom still is, especially the interest in old Doom/Doom II. I grew up with that game and I would have never thought that I still enjoy those episodes more than 20 years later. I played the PSX version first, around 1995, and was "shocked" how different the PC version turned out to be. But it was worth it. I'm glad to have learned about DB, Slade etc. and I'm trying to work on my own episode like many others did.
I think you need void(deleted area) around doors. I had same thing when I did my first maps.
I think I heard a door sound. Did you accidentally make that wall a door?
My very first map already included lifts and teleports and even three little scripts and it worked out fine. I suppose I have to scrap that area in the worst case and try a new one. In the meanwhile I can work on sth. else..
If you really want a definitive answers then you should make your map available for perusal and analysis.
Else you just invite a round of guesses.
YEAH, that was probably part of the problem. That helped a lot. I also found two incomplete sectors and some bad texture alignments.
Thank you for your hint about that !!


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A tactical air navigation system, commonly referred to by the acronym TACAN, is a navigation system used by military aircraft. It provides the user with bearing and distance (slant-range or hypotenuse) to a ground or ship-borne station. It is a more accurate version of the VOR/DME system that provides bearing and range information for civil aviation. The DME portion of the TACAN system is available for civil use; at VORTAC facilities where a VOR is combined with a TACAN, civil aircraft can receive VOR/DME readings. Aircraft equipped with TACAN avionics can use this system for en route navigation as well as non-precision approaches to landing fields. The Space Shuttle is one such vehicle that was designed to use TACAN navigation but later upgraded with GPS as a replacement.

The typical TACAN onboard user panel has control switches for setting the channel (corresponding to the desired surface station's assigned frequency), the operation mode for either transmit/receive (T/R, to get both bearing and range) or receive only (REC, to get bearing but not range). Capability was later upgraded to include an air-to-air mode (A/A) where two airborne users can get relative slant-range information. Depending on the installation, Air-to-Air mode may provide range, closure (relative velocity of the other unit), and bearing, though an air-to-air bearing is noticeably less precise than a ground-to-air bearing. A TACAN only equipped aircraft cannot receive bearing information from a VOR only station.

History

The TACAN navigation system is an evolution of radio transponder navigation systems that date back to the British Oboe system of World War II. In the United States, many companies were involved with the development of TACAN for military aircraft. Hoffman Laboratories Div. of the Hoffman Electronics Corp.–Military Products Division (now NavCom Defense Electronics) was a leader in developing the present TACAN system in the US starting in the late 1950s.

Operation
TACAN in general can be described as the military version of the VOR/DME system. It operates in the frequency band 960-1215 MHz. The bearing unit of TACAN is more accurate than a standard VOR since it makes use of a two-frequency principle, with 15 Hz and 135 Hz components, and because UHF transmissions are less prone to signal bending than VHF.

The distance measurement component of TACAN operates with the same specifications as civil DMEs. Therefore, to reduce the number of required stations, TACAN stations are frequently co-located with VOR facilities. These co-located stations are known as VORTACs. This is a station composed of a VOR for civil bearing information and a TACAN for military bearing information and military/civil distance measuring information. The TACAN transponder performs the function of a DME without the need for a separate co-located DME. Because the rotation of the antenna creates a large portion of the azimuth (bearing) signal, if the antenna fails, the azimuth component is no longer available and the TACAN downgrades to a DME only mode.

Accuracy

Theoretically a TACAN should provide a 9-fold increase in accuracy compared to a VOR, but operational use has shown only an approximate 3-fold increase.

Accuracy of the 135 Hz azimuth component is ±1° or ±63 m at 3.75 km. Accuracy of the DME portion must be  or 3 percent of slant range distance, whichever is greater, per FAA 9840.1 1982. and FAA N8200.121

TACAN stations can provide distance up to 390 nautical miles.

Modern TACANs are much more accurate. The requirement now is to have portable TACAN that is IFR certifiable, both station and portable systems. The latest modern version of TACAN has been tested and could be a feasible back-up to future Air traffic control systems and may even be integrated into systems for a seamless back up.

Past TACANs have relied on high output power (up to 10,000 watts) to ensure good signal in space to overcome nulls present in antenna design and to provide their required 200 mile range. With the advancement of technology, antenna design has improved with higher gain antennas, much shallower nulls, and lighter construction. Now it's feasible to have a 200 nmi range with a 400 watt TACAN DME transmitter, making the TACAN package much smaller, more portable and more reliable (a decrease in power also reduces heat, which lengthens the life of electronics).

On the first Space Shuttle flight, Capcom Joseph P. Allen reported up to the crew that their TACANs had locked onto the Channel 111 signals at St. Petersburg, FL at a range of 250 miles.

TACAN is getting smaller: full TACAN coverage can now be provided in a system that can be carried on a single trailer weighing less than 4000 lbs, and set up by two people in less than an hour. TACAN Transceivers can now be as small as lunch boxes (with full coverage and range) and the antennas can be reduced from 800 pounds to less than 100 pounds.

Benefits

Because the azimuth and range units are combined in one system it provides for simpler installation. Less space is required than a VOR because a VOR requires a large counterpoise and a fairly complex phased antenna system. A TACAN system theoretically might be placed on a building, a large truck, an airplane, or a ship, and be operational in a short period of time. An airborne TACAN receiver can be used in air-to-air mode, which allows two cooperating aircraft to find their relative bearings and distance.

Drawbacks
For military usage a primary drawback is lack of the ability to control emissions (EMCON) and stealth. Naval TACAN operations are designed so an aircraft can find the ship and land. There is no encryption involved, an enemy can simply use the range and bearing provided to attack a ship equipped with a TACAN. Some TACANs have the ability to employ a "Demand Only" mode wherein they will only transmit when interrogated by an aircraft on-channel. It is likely that TACAN will be replaced with a differential GPS system similar to the Local Area Augmentation System called JPALS. The Joint Precision Approach and Landing System has a low probability of intercept to prevent enemy detection and an aircraft carrier version can be used for autoland operations.

Some systems used in the United States modulate the transmitted signal by using a 900 RPM rotating antenna. Since this antenna is fairly large and must rotate 24 hours a day, it can cause reliability issues. Modern systems have antennas that use electronic rotation (instead of mechanical rotation) with no moving parts.

See also
Battle of Lima Site 85 (SAC TACAN captured March 1968)
Distance measuring equipment
Electronics technician
Global Positioning System
VHF omnidirectional range
Wide Area Augmentation System

References

External links

dB Systems, Inc. - Manufacturer of mechanically scanned, electronically scanned, shipboard, man-portable, and tactical TACAN Antennas - Complete TACAN Antenna profile with datasheets and photos
Rantec Microwave Systems - Manufacturer of non-rotating TACAN antennas - Complete with antenna internal photos and specs
Moog Navigation and Surveillance Systems - Fixed site, shipboard, mobile and man-portable TACAN systems
Leonardo Air Traffic Management - Fixed site TACAN systems

Avionics
Radio navigation
Aircraft instruments
Air navigation

redpajama_set_name: RedPajamaWikipedia


text:
Book article / Book chapter (5)
Physiologische Chemie (43)
Schwertkärpfling (18)
Krebs <Medizin> (4)
Onkogen (4)
Xiphophorus (4)
Lebendgebärende Zahnkarpfen (3)
Protein-Tyrosin-Kinasen (3)
Schartl, Manfred (108) (remove)
Theodor-Boveri-Institut für Biowissenschaften (108) (remove)
108 search hits
Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements (2018)
Schartl, Manfred ; Schories, Susanne ; Watamatsu, Yuko ; Nagao, Yusuke ; Hashimoto, Hisashi ; Bertin, Chloé ; Mourot, Brigitte ; Schmidt, Cornelia ; Wilhelm, Dagmar ; Centanin, Lazaro ; Guiguen, Yann ; Herpin, Amaury
Background: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. Results: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. Conclusions: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.
The colorful sex chromosomes of teleost fish (2018)
Kottler, Verena A. ; Schartl, Manfred
Teleost fish provide some of the most intriguing examples of sexually dimorphic coloration, which is often advantageous for only one of the sexes. Mapping studies demonstrated that the genetic loci underlying such color patterns are frequently in tight linkage to the sex-determining locus of a species, ensuring sex-specific expression of the corresponding trait. Several genes affecting color synthesis and pigment cell development have been previously described, but the color loci on the sex chromosomes have mostly remained elusive as yet. Here, we summarize the current knowledge about the genetics of such color loci in teleosts, mainly from studies on poeciliids and cichlids. Further studies on these color loci will certainly provide important insights into the evolution of sex chromosomes.
A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths (2018)
Biscotti, Maria Assunta ; Adolfi, Mateus Contar ; Barucca, Marco ; Forconi, Mariko ; Pallavicini, Alberto ; Gerdol, Marco ; Canapa, Adriana ; Schartl, Manfred
Gonadal sex differentiation and reproduction are the keys to the perpetuation of favorable gene combinations and positively selected traits. In vertebrates, several gonad development features that differentiate tetrapods and fishes are likely to be, at least in part, related to the water-to-land transition. The collection of information from basal sarcopterygians, coelacanths, and lungfishes, is crucial to improve our understanding of the molecular evolution of pathways involved in reproductive functions, since these organisms are generally regarded as "living fossils" and as the direct ancestors of tetrapods. Here, we report for the first time the characterization of >50 genes related to sex differentiation and gametogenesis in Latimeria menadoensis and Protopterus annectens. Although the expression profiles of most genes is consistent with the intermediate position of basal sarcopterygians between actinopterygian fish and tetrapods, their phylogenetic placement and presence/absence patterns often reveal a closer affinity to the tetrapod orthologs. On the other hand, particular genes, for example, the male gonad factor gsdf (Gonadal Soma-Derived Factor), provide examples of ancestral traits shared with actinopterygians, which disappeared in the tetrapod lineage.
Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates (2016)
Brunet, Frédéric G. ; Volff, Jean-Nicolas ; Schartl, Manfred
The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling.
Germ cell and tumor associated piRNAs in the medaka and \(Xiphophorus\) melanoma models (2016)
Kneitz, Susanne ; Mishra, Rasmi R. ; Chalopin, Domitille ; Postlethwait, John ; Warren, Wesley C. ; Walther, Ronald B. ; Schartl, Manfred
Background A growing number of studies report an abnormal expression of Piwi-interacting RNAs (piRNAs) and the piRNA processing enzyme Piwi in many cancers. Whether this finding is an epiphenomenon of the chaotic molecular biology of the fast dividing, neoplastically transformed cells or is functionally relevant to tumorigenesisis is difficult to discern at present. To better understand the role of piRNAs in cancer development small laboratory fish models can make a valuable contribution. However, little is known about piRNAs in somatic and neoplastic tissues of fish. Results To identify piRNA clusters that might be involved in melanoma pathogenesis, we use several transgenic lines of medaka, and platyfish/swordtail hybrids, which develop various types of melanoma. In these tumors Piwi, is expressed at different levels, depending on tumor type. To quantify piRNA levels, whole piRNA populations of testes and melanomas of different histotypes were sequenced. Because no reference piRNA cluster set for medaka or Xiphophorus was yet available we developed a software pipeline to detect piRNA clusters in our samples and clusters were selected that were enriched in one or more samples. We found several loci to be overexpressed or down-regulated in different melanoma subtypes as compared to hyperpigmented skin. Furthermore, cluster analysis revealed a clear distinction between testes, low-grade and high-grade malignant melanoma in medaka. Conclusions Our data imply that dysregulation of piRNA expression may be associated with development of melanoma. Our results also reinforce the importance of fish as a suitable model system to study the role of piRNAs in tumorigenesis.
Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka (2016)
Adolfi, Mateus C. ; Herpin, Amaury ; Regensburger, Martina ; Sacquegno, Jacopo ; Waxman, Joshua S. ; Schartl, Manfred
In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage.
A transcriptome derived female-specific marker from the invasive Western mosquitofish (Gambusia affinis) (2015)
Lamatsch, Dunja K. ; Adolfsson, Sofia ; Senior, Alistair M. ; Christiansen, Guntram ; Pichler, Maria ; Ozaki, Yuichi ; Smeds, Linnea ; Schartl, Manfred ; Nakagawa, Shinichi
Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3' UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.
Whole body melanoma transcriptome response in medaka (2015)
Schartl, Manfred ; Shen, Yingjia ; Maurus, Katja ; Walter, Ron ; Tomlinson, Chad ; Wilson, Richard K. ; Postlethwait, John ; Warren, Wesley C.
The incidence of malignant melanoma continues to increase each year with poor prognosis for survival in many relapse cases. To reverse this trend, whole body response measures are needed to discover collaborative paths to primary and secondary malignancy. Several species of fish provide excellent melanoma models because fish and human melanocytes both appear in the epidermis, and fish and human pigment cell tumors share conserved gene expression signatures. For the first time, we have examined the whole body transcriptome response to invasive melanoma as a prelude to using transcriptome profiling to screen for drugs in a medaka (Oryzias latipes) model. We generated RNA-seq data from whole body RNA isolates for controls and melanoma fish. After testing for differential expression, 396 genes had significantly different expression (adjusted p-value <0.02) in the whole body transcriptome between melanoma and control fish; 379 of these genes were matched to human orthologs with 233 having annotated human gene symbols and 14 matched genes that contain putative deleterious variants in human melanoma at varying levels of recurrence. A detailed canonical pathway evaluation for significant enrichment showed the top scoring pathway to be antigen presentation but also included the expected melanocyte development and pigmentation signaling pathway. Results revealed a profound down-regulation of genes involved in the immune response, especially the innate immune system. We hypothesize that the developing melanoma actively suppresses the immune system responses of the body in reacting to the invasive malignancy, and that this mal-adaptive response contributes to disease progression, a result that suggests our whole-body transcriptomic approach merits further use. In these findings, we also observed novel genes not yet identified in human melanoma expression studies and uncovered known and new candidate drug targets for further testing in this malignant melanoma medaka model.
Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae (2015)
Adolfi, Mateus C. ; Carreira, Ana C. O. ; Jesus, Lázaro W. O. ; Bogerd, Jan ; Funes, Rejane M. ; Schartl, Manfred ; Sogayar, Mari C. ; Borella, Maria I.
Background The dmrt1 and sox9 genes have a well conserved function related to testis formation in vertebrates, and the group of fish presents a great diversity of species and reproductive mechanisms. The lambari fish (Astyanax altiparanae) is an important Neotropical species, where studies on molecular level of sex determination and gonad maturation are scarce. Methods Here, we employed molecular cloning techniques to analyze the cDNA sequences of the dmrt1 and sox9 genes, and describe the expression pattern of those genes during development and the male reproductive cycle by qRT-PCR, and related to histology of the gonad. Results Phylogenetic analyses of predicted amino acid sequences of dmrt1 and sox9 clustered A. altiparanae in the Ostariophysi group, which is consistent with the morphological phylogeny of this species. Studies of the gonad development revealed that ovary formation occurred at 58 days after hatching (dah), 2 weeks earlier than testis formation. Expression studies of sox9 and dmrt1 in different tissues of adult males and females and during development revealed specific expression in the testis, indicating that both genes also have a male-specific role in the adult. During the period of gonad sex differentiation, dmrt1 seems to have a more significant role than sox9. During the male reproductive cycle dmrt1 and sox9 are down-regulated after spermiation, indicating a role of these genes in spermatogenesis. Conclusions For the first time the dmrt1 and sox9 were cloned in a Characiformes species. We show that both genes have a conserved structure and expression, evidencing their role in sex determination, sex differentiation and the male reproductive cycle in A. altiparanae. These findings contribute to a better understanding of the molecular mechanisms of sex determination and differentiation in fish.
Beyond the zebrafish: diverse fish species for modeling human disease (2014)
Schartl, Manfred
In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research.
A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon (2014)
Tomaszkiewicz, Marta ; Chalopin, Domitille ; Schartl, Manfred ; Galiana, Delphine ; Volff, Jean-Nicolas
Background: Teleost fish present a high diversity of sex determination systems, with possible frequent evolutionary turnover of sex chromosomes and sex-determining genes. In order to identify genes involved in male sex determination and differentiation in the platyfish Xiphophorus maculatus, bacterial artificial chromosome contigs from the sex-determining region differentiating the Y from the X chromosome have been assembled and analyzed. Results: A novel three-copy gene called teximY (for testis-expressed in Xiphophorus maculatus on the Y) was identified on the Y but not on the X chromosome. A highly related sequence called texim1, probably at the origin of the Y-linked genes, as well as three more divergent texim genes were detected in (pseudo) autosomal regions of the platyfish genome. Texim genes, for which no functional data are available so far in any organism, encode predicted esterases/lipases with a SGNH hydrolase domain. Texim proteins are related to proteins from very different origins, including proteins encoded by animal CR1 retrotransposons, animal platelet-activating factor acetylhydrolases (PAFah) and bacterial hydrolases. Texim gene distribution is patchy in animals. Texim sequences were detected in several fish species including killifish, medaka, pufferfish, sea bass, cod and gar, but not in zebrafish. Texim-like genes are also present in Oikopleura (urochordate), Amphioxus (cephalochordate) and sea urchin (echinoderm) but absent from mammals and other tetrapods. Interestingly, texim genes are associated with a Helitron transposon in different fish species but not in urochordates, cephalochordates and echinoderms, suggesting capture and mobilization of an ancestral texim gene in the bony fish lineage. RT-qPCR analyses showed that Y-linked teximY genes are preferentially expressed in testis, with expression at late stages of spermatogenesis (late spermatids and spermatozeugmata). Conclusions: These observations suggest either that TeximY proteins play a role in Helitron transposition in the male germ line in fish, or that texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.
Novel Method for Analysis of Allele Specific Expression in Triploid Oryzias latipes Reveals Consistent Pattern of Allele Exclusion (2014)
Garcia, Tzintzuni I. ; Matos, Isa ; Shen, Yingjia ; Pabuwal, Vagmita ; Coelho, Maria Manuela ; Wakamatsu, Yuko ; Schartl, Manfred ; Walter, Ronald B.
Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.
The MAPK pathway as an apoptosis enhancer in melanoma (2014)
Haydn, Johannes M. ; Hufnagel, Anita ; Grimm, Johannes ; Maurus, Katja ; Schartl, Manfred ; Meierjohann, Svenja
Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAFV600E–mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAFV600E-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis.
p53 Gene Targeting by Homologous Recombination in Fish ES Cells (2013)
Yan, Yan ; Hong, Ni ; Chen, Tiansheng ; Li, Mingyou ; Wang, Tiansu ; Guan, Guijun ; Qiao, Yongkang ; Chen, Songlin ; Schartl, Manfred ; Li, Chang-Ming ; Hong, Yunhan
Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.
Divergent Expression Regulation of Gonad Development Genes in Medaka Shows Incomplete Conservation of the Downstream Regulatory Network of Vertebrate Sex Determination (2013)
Herpin, Amaury ; Adolfi, Mateus C. ; Nicol, Barbara ; Hinzmann, Maria ; Schmidt, Cornelia ; Klughammer, Johanna ; Engel, Mareen ; Tanaka, Minoru ; Guiguen, Yann ; Schartl, Manfred
Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.
Characterization of Sex Determination and Sex Differentiation Genes in Latimeria (2013)
Forconi, Mariko ; Canapa, Adriana ; Barucca, Marco ; Biscotti, Maria A. ; Capriglione, Teresa ; Buonocore, Francesco ; Fausto, Anna M. ; Makapedua, Daisy M. ; Pallavicini, Alberto ; Gerdol, Marco ; De Moro, Gianluca ; Scapigliati, Giuseppe ; Olmo, Ettore ; Schartl, Manfred
Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.
Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells (2013)
Schul, Daniela ; Schmitt, Alexandra ; Regneri, Janine ; Schartl, Manfred ; Wagner, Toni Ulrich
Bone Morphogenetic Proteins (BMPs) are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells.
Comprehensive phylogenetic analysis of all species of swordtails and platies (Pisces: Genus Xiphophorus) uncovers a hybrid origin of a swordtail fish, Xiphophorus monticolus, and demonstrates that the sexually selected sword originated in the ancestral lineage of the genus, but was lost again secondarily (2013)
Kang, Ji Hyoun ; Schartl, Manfred ; Walter, Ronald B. ; Meyer, Axel
Background: Males in some species of the genus Xiphophorus, small freshwater fishes from Meso-America, have an extended caudal fin, or sword - hence their common name "swordtails". Longer swords are preferred by females from both sworded and - surprisingly also, non-sworded (platyfish) species that belong to the same genus. Swordtails have been studied widely as models in research on sexual selection. Specifically, the pre-existing bias hypothesis was interpreted to best explain the observed bias of females in presumed ancestral lineages of swordless species that show a preference for assumed derived males with swords over their conspecific swordless males. However, many of the phylogenetic relationships within this genus still remained unresolved. Here we construct a comprehensive molecular phylogeny of all 26 known Xiphophorus species, including the four recently described species (X. kallmani, X. mayae, X. mixei and X. monticolus). We use two mitochondrial and six new nuclear markers in an effort to increase the understanding of the evolutionary relationships among the species in this genus. Based on the phylogeny, the evolutionary history and character state evolution of the sword was reconstructed and found to have originated in the common ancestral lineage of the genus Xiphophorus and that it was lost again secondarily. Results: We estimated the evolutionary relationships among all known species of the genus Xiphophorus based on the largest set of DNA markers so far. The phylogeny indicates that one of the newly described swordtail species, Xiphophorus monticolus, is likely to have arisen through hybridization since it is placed with the southern platyfish in the mitochondrial phylogeny, but with the southern swordtails in the nuclear phylogeny. Such discordance between these two types of markers is a strong indication for a hybrid origin. Additionally, by using a maximum likelihood approach the possession of the sexually selected sword trait is shown to be the most likely ancestral state for the genus Xiphophorus. Further, we provide a well supported estimation of the phylogenetic relationships between the previously unresolved northern swordtail groups. Conclusions: This comprehensive molecular phylogeny of the entire genus Xiphophorus provides evidence that a second swordtail species, X. monticolus, arose through hybridization. Previously, we demonstrated that X. clemenciae, another southern swordtail species, arose via hybridization. These findings highlight the potential key role of hybridization in the evolution of this genus and suggest the need for further investigations into how hybridization contributes to speciation more generally.
Conserved expression signatures between medaka and human pigment cell tumors (2012)
Schartl, Manfred ; Kneitz, Susanne ; Wilde, Brigitta ; Wagner, Toni ; Henkel, Christiaan V. ; Spaink, Hermann P. ; Meierjohann, Svenja
Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.
Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model (2012)
Menescal, Luciana ; Schmidt, Cornelia ; Liedtke, Daniel ; Schartl, Manfred
Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifeninducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.
CrossQuery : A Web Tool for Easy Associative Querying of Transcriptome Data (2011)
Wagner, Toni U. ; Fischer, Andreas ; Thoma, Eva C. ; Schartl, Manfred
Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deepsequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.
CrossQuery: A Web Tool for Easy Associative Querying of Transcriptome Data (2011)
Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deep-sequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.
Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid \(Squalius\) \(alburnoides\) (2011)
Matos, Isa ; Sucena, Èlio ; Machado, Miguel P ; Gardner, Rui ; Inácio, Ângela ; Schartl, Manfred ; Coelho, Maria M
Background Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome) and males of an unknown Anaecypris hispanica- like species (A genome). S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition) silencing of one of the three alleles (mainly of the P allele) occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen. In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns. Results To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range. Conclusions Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where S. pyrenaicus is sympatric with S. alburnoides. We discuss mechanisms that could lead to the formation of mosaic S. alburnoides and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids."
Highly Asynchronous and Asymmetric Cleavage Divisions Accompany Early Transcriptional Activity in Pre-Blastula Medaka Embryos (2011)
Kraeussling, Michael ; Wagner, Toni Ulrich ; Schartl, Manfred
In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation (2010)
Meierjohann, Svenja ; Hufnagel, Anita ; Wende, Elisabeth ; Kleinschmidt, Markus A. ; Wolf, Katarina ; Friedl, Peter ; Gaubatz, Stefan ; Schartl, Manfred
Background: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTKdriven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.
Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins (2010)
Teutschbein, Janka ; Haydn, Johannes M. ; Samans, Birgit ; Krause, Michael ; Eilers, Martin ; Schartl, Manfred ; Meierjohann, Svenja
Background: Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods: Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results: Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion: Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.
Lineage-specific co-evolution of the Egf receptor/ligand signaling system (2010)
Laisney, Juliette A. G. C. ; Braasch, Ingo ; Walter, Ronald B. ; Meierjohann, Svenja ; Schartl, Manfred
Background: The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. Results: We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Conclusions: Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system.
Neurotrophin-6 is a new member of the nerve growth factor family (1994)
Götz, Rudolf ; Köster, Reinhard ; Winkler, Christoph ; Raulf, Friedrich ; Lottspeich, Friedrich ; Schartl, Manfred ; Thoenen, Hans
DURING vertebrale development, many neurons depend for survival and differentiation on their target cells\(^{1-3}\). The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF)\(^1\). NGF and the other known members of tbe neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT -3) and neurotrophin-4/5 (NT -4/5) are conserved as distinct genes over large evolutionary distances\(^{4 -6}\). Here we report the cloning of neurotrophin-6 (NT -6), a new member of this family from the teleost fish Xiphophorus. NT -6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT -6 from cell surface and extracellular matrix molecules. Recombinant purified NT -6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT -6 is expressed in tbe embryonie valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecuJes may modulate its action in the nervous system .
Autocrine stimulation of the Xmrk receptor tyrosine kinase in Xiphophorus melanoma cells and identification of a source for the physiological ligand (1994)
Malitschek, Barbara ; Wittbrodt, Joachim ; Fischer, Petra ; Lammers, Reiner ; Ullrich, Axel ; Schartl, Manfred
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
Recurrent origin of a sexually selected trait in Xiphophorus fishes inferred from a molecular phylogeny (1994)
Meyer, Axel ; Morrissey, Jean M. ; Schartl, Manfred
DARWIN\(^1\) believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes\(^{2,3}\). Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondrial and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the cvolution of this sexually selected trait.
Male polymorphism in Limia perugiae (Pisces: Poeciliidae) (1994)
Erbelding-Denk, Claudia ; Schroder, Johannes H. ; Schartl, Manfred ; Nanda, Indrajit ; Schmid, Michael ; Epplen, Jörg T.
The male-polymorphic poeciliid fish, Limia perugiae, a small teleostean endemic to the southeast of the Caribbean island Hispafiola, consists of three male size morphs with uniform females. Large males differentiate at a size va:rying between 25 and 38 mm; intermediate males, between 21 and 25 mm. Under competition, !arge males exhibit an elaborate courtship display, whereas small males show only a sneak-chase behavior. Intermediate males adapt their tactics to the respective competitors. However, all malemorphs can switch from courtship display to sneak-chase behavior. In large mating groups with four males of different size and five or six virgin females, large dominant a-males as weil as small subordinate \(\delta\)-males did not produce any offspring. Unexpectedly, all progeny were sired exclusively by the intemediate subordinate ß- and \(\gamma\)-males. Breeding experiments with the three male morphs can best be explained by a model of Y -linked genes for small and !arge size which are both suspended by the activity of an autosomal recessive repressor responsible for the development of intermediate males. The dominant allele of the recessive repressor, in either its homoorits heterozygous state, activates the Y-chromosomal genes for !arge or small size, respectively. Accordingly, intermediate males may produce male offspring of all size classes, depending on the presence of either the Y-linked gene or the autosomal repressor.
Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus (1994)
Nakayama, Ichiro ; Foresti, Fausto ; Tewari, Rita ; Schartl, Manfred ; Chourrout, Daniel
In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA ofmales and females ofthe rainbow trout (XX/XY) and of Leporinus elongatus (ZW /ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinusfriderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.
Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene (1994)
Winkler, Christoph ; Wittbrodt, Joachim ; Lammers, Reiner ; Ullrich, Axel ; Schartl, Manfred
Xmrk encodes a subclass 1 receptor tyrosine kinase (RTK) which has been cloned from the melanomainducing locus Tu of the poeciliid fish Xiphophorus. To demonstrate a high oncogenic potential in vivo we transferred the gene into early embryos of the closely related medakafish. Ectopic expression of the Xmrk oncogene under the control of a strong, constitutive promoter (CMVTk) led to the induction of embryonic tumors with high incidence, after short latency periods, and with a specific pattern of affected tissues. We demonstrate ligand-dependent transformation in vivo using a chimeric receptor consisting of the extracellular and transmembrane domains of the human EGF receptor (HER) and the cytoplasmatic domain of Xmrk. Expression of the chimeric receptor alone does not lead to ldnase activation or induction of tumors. Coexpression of the chimera with its corresponding ligand, human transforming growth factor alpha (bTGF(X), however, results in the activation of the chimeric RTK. In injected fish embryos the induction of the neoplastic growth is observed with similar incidence and tissue distribution as in embryos carrying the native Xmrk oncogene suggesting that the ligand as well as factors downstream of tbe RTK are required for tumor formation. In this study we show single-step induction of tumors by ectopic expression of RTKs in vivo substantiating tbe significance of autocrine stimulation in RTK induced tumors in vertebrales.
Molecular approaches to study the genetic regulation of the fish reproductive system (1994)
Schreibman, Martin P. ; Schartl, Manfred ; Kallman, Klaus D. ; Magliulo-Cepriano, Lucia
Genetics and molecular biology of tumour formation in Xiphophorus (1994)
Altschmied, Joachim ; Schartl, Manfred
Evolutionary origin and molecular biology of the melanoma-inducing oncogene of Xiphophorus (1994)
Schartl, A. ; Dimitrijevic, N. ; Schartl, Manfred
The conservation of neurotrophic factors during vertebrate evolution (1994)
Gotz, R. ; Schartl, Manfred
Methodik und Anwendungsgebiete des genetischen Fingerabdruckverfahrens (1994)
Lubjuhn, T. ; Schartl, Manfred ; Epplen, J. T.
Different expression patterns of oncogenes and proto-oncogenes in hereditary and carcinogen-induced tumors of Xiphophorus (1993)
Mäueler, Winfried ; Schartl, Angelika ; Schartl, Manfred
Hereditary melanoma in Xiphophorus hybrids canying the melanoma·induclng Tu-Sd locus is caused by transcriptional activation of the Xmrk gene that resides at the Tu·Sd locus and encodes a novel member of receptor tyrosine kinases (RTK). ln this study, a total of 17 hereditary melanomas from various hybrid genotypes harbouring 7 different Tu alleles were also found to aver-express the correspondlng Xmrlc alleles. The Ievei of over-expression correlated with the degree of malignancy of the melanoma. ln addition, Xsrc expression was high ln many malignant melanomas. Expression pattems and Ieveis of the Xiphophorus EGF-receptor gene (Xerb B), the c-myc (Xmyc), and the PDGF (Xsls) gene(s) were not intriguing. Transcription of the ras gene(s) may be correlated to secondary events of melanoma progression. Expression pattems of Xfms, the Xiphophorus CSF-1 receptor homologue, can be explained by different contents of infiltrating macrophages in the tumors. ln carcinogen-induced tumors includlng one melanoma no significant expression of the Xmrk oncogene could be detected. Xsrc expression, however, was strikingly high. This indicates that activation of oncogenes other than Xmrk ls instrumental in tumorigenesls of neoplasia of non-hereditary origin.
Which home for coelacanth? (1993)
Schliewen, U. ; Fricke, H. ; Schartl, Manfred ; Epplen, Jörg T. ; Paabo, S.
High mating success of low rank males in Limia perugiae (Pisces: Poeciliidae) as determined by DNA-fingerprinting (1993)
Schartl, Manfred ; Erbelding-Denk, C. ; Hölter, S. ; Nanda, I. ; Schmid, M. ; Schröder, J. H. ; Epplen, J. T.
Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behaviour and access to the females. In general social dominance requires large investments which in turn may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups due to the difficulties of determining paternity by classical methods using non-molecular markers. DNA fingerprinting overcomes these problems offering the possibility to determine genetic relationships and mating patterns within larger groups. Using this approach we have recently shown (Schartl et al., 1993) that in the poeciliid fish Limia perugiae in small mating groups the dominant male has 100% mating success, while in larger groups its contribution to the offspring unexpectedly drops to zero. The reproductive failure under such social conditions is explained by the inability of the ex-male to protect all the females simultaneously against mating attempts of his numerous subordinate competitors.
Oncogenes and melanoma formation in Xiphoporus (Teleostei: Poeciliidae) (1993)
Schartl, Manfred ; Wittbrodt, J. ; Mäueler, W. ; Raulf, F. ; Adam, D. ; Hannig, G. ; Telling, A. ; Storch, F. ; Andexinger, S. ; Robertson, S. M.
In Xiphophorus melanoma formation has been attributed by classical genetic findings to the overexpression of a cellular oncogene (Tu) due to elimination of the corresponding regulatory gene locus in hybrids. We have attempted to elucidate this phenomenon on the molecular biological level. Studies on the structure and expression of known proto-oncogenes revealed that several of these genes, especially the c-src gene of Xiphophorus, may act as effectors in establishing the neoplastic phenotype of the melanoma cells . However, these genes appear more to participate in secondary steps of tumorigenesis. Another gene, being termed Xmrk, which represents obviously a so far unknown proto-oncogene but with a cons iderably high similarity to the epidermal growth-factorreceptor gene, was mapped to the Tu-containing region of the chromosome. This gene shows features with respect to its structure and expression that seem to justify it to be regarded as a candidate for a gene involved in the primary processes leading to neoplastic transformation of pigment cells in Xiphophorus.
Effect of growth hormone on the growth rate of the gilthead seabream (Sparus aurata), and use of different constructs for the production of transgenic fish (1993)
Cavari, Benzion ; Funkenstein, Bruria ; Chen, Thomas T. ; Gonzalez-Villasenor, Lucia Irene ; Schartl, Manfred
When bovine or human growth hormones (GH) were injected into 6 months old (about 10 g) gilthead seabream, the growth rate of the fish, as measured by changes in their weight, increased by only about 15% compared with the saline-injected control. No effect or even slight inhibition of the growth rate was obtained when chicken or porcine GHs were injected. In a preliminary experiment, it was found that injection ofthe native GH increased the growth rate ofthe fish by about 20% after treatment for only 2 weeks. An expression vector, using the pRE1 plasmid and transformation into MZl cells, produced the gilthead seabream GH, providing a supply for further experiments on the effect of the homologaus GH on growth. Two reporter genes, ß-galactosidase (lacZ) and melanoma oncogene of Xiphophorus (mrk YY), were microinjected into fertilized eggs of S. aurata. Expression of these two genes could be demonstrated in 2-day-old embryos, the lacZ gene by staining of its enzymatic product, and the mrk YY gene by its phenotypic expression.
All-fish gene constructs for growth hormone gene transfer in fish (1993)
Cavari, Benzion ; Hong, Yunhan ; Funkenstein, Bruria ; Moav, Boaz ; Schartl, Manfred
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
Sequence of the growth hormone (GH) gene from the silver carp (Hypophthalmichthys molitrix) and evolution of GH genes in vertebrates (1993)
Hong, Yunhan ; Schartl, Manfred
The silver carp (Hypophthalmichthys molitrix) growth hormone (GH) genewas isolated and sequenced following amplification from genomic DNA by the polymerase chain reaction. The gene spans a region of approx. 2.5 kb nucleotides (nt) and consists of five exons. The sequence predicts a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 hydrophobic aa residues. The arrangement of exons and introns is identical to the GH genes of common carp, grass carp, and very similar to mammals and birds, but quite different from that for the GH genes of tilapia and salmonids. The silver carp GH gene shares a high homology at the nt and aa Ievels with those of grass carp (95.3% nt, 99.5% aa) and of common carp (81% nt, 95.7% aa).
Tumor suppression in Xiphophorus by an accidentally acquired promoter (1993)
Adam, Dieter ; Dimitrijevic, Nicola ; Schartl, Manfred
Melanoma formation in the teleost Xiphophorus is caused by a dominant genetic locus, Tu. This locus includes the Xmrk oncogene, which encodes a receptor tyrosine kinase. Tumor induction is. suppressed in wild-type fish by a tumor suppressor locus, R. Molecular genetic analyses revealed that the Tu locus emerged by nonhomologaus recombination of the Xmrk proto-oncogene with a previously uncharacterized sequence, D. This event generated an additional copy of Xmrk with a new promoter. Suppression of the new Xmrk promoter by R in parental fish and its deregulation in hybrids explain the genetics of melanoma formation in Xiphophorus.
Reproductive failure of dominant males in the poeciliid fish Limia perugiae determined by DNA fingerprinting (1993)
Schartl, Manfred ; Erbelding-Denk, Claudia ; Holter, Sabine ; Nanda, Indrajit ; Schmid, Michael ; Schroder, Johannes H. ; Epplen, Jörg T.
Hierarchical structures among male indlviduals in a population are frequently reflected ln differences in aggressive and reproductive behavior and access to the females. In general, sodal dominance requires the Investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesls has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Borke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poecUiid fish Limia perugitu, in small matlng groups the dominant male has 8 mating success of 100%, whereas ln larger groups lts contribution to the offspring unexpectedly drops to zero.
Primitive sex chromosomes in poeciliid fishes harbor simple repetitive DNA sequences (1993)
Nanda, Indrajit ; Schartl, Manfred ; Epplen, Jörg T. ; Feichtinger, Wolfgang ; Schmid, Michael
The demonstration ofthe chromosomal mode ofsex determinationvia genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.
Development of a heavy metal-inducible fish-specific expression vector for gene transfer in vitro and in vivo (1993)
Hong, Yunhan ; Winkler, Christoph ; Brem, Gottfried ; Schartl, Manfred
The promoter of the rainbow trout metallothionein B gene ( tMTb) was isolated from genomic DNA by the polymerase chain reaction (PCR ), fused to the bacterial chloramphenicol acetyltransferase (CAT) genein an expression vector, and functionally analyzed in one human cellline and four fish celllines. This promoter exhibited an extremely low basal expression in all celllines and was zincand cadmium-inducible except in the fish melanoma cell line where the promoter was completely inactive. The metal-induced expression patterns were cellline-specific. In general the fish promoter was more species- and cell type-specific than its human counterpart. In a transient assay it was functional in developing embryos of the medaka ( Oryzias /atipes). These properties make this promoter suitable for inducible, tissue-specific expression of transgenes and for in vivo studies of gene function and regulation.


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