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The Russian Reader
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PSA (It's Time to Start the Deprogramming) January 17, 2021
Fifty Members of the Russian Academy of Sciences: "We Urge the Court to Release Azat Miftakhov" January 16, 2021
Kalinka Malinka January 15, 2021
Zinaida Pozdnyakova, "Kalinin Prospekt" January 10, 2021
They Have Nothing Better to Do January 9, 2021
Political Prisoner Dmitry Pchelintsev: "Please Tell Mom That I'm Well" January 8, 2021
A Letter to the International Congress of Mathematicians on the Azat Miftakhov Case January 4, 2021
Love Conquers All January 3, 2021
Copyleft January 3, 2021
David Frenkel: The Year 2020 in Pictures January 1, 2021
Arkady Kots: We Will Have to Fight for the Future December 30, 2020
Russian Justice Ministry Adds Five New "Foreign Agents" to Its List December 28, 2020
Stopping Foreign Agents, Killing Russian Education December 28, 2020
How I Was Friends with Billionaires December 23, 2020
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Tag: Alexei Ulyukayev
Olga Romanova: How "Law Enforcement" Works in Russia
Michael Calvey in court. Photo by Maxim Shemetov. Courtesy of Reuters and Republic
"We Give You Serebrennikov and You Give us Calvey": How Law Enforcement Works
Olga Romanova
"Who would make the decision about your arrest?"
"My colleagues would betray me, but they would vet it with my bosses."
"What about Vasya [a big businessman]?"
"Cops, the economic security squad. It's enough for the word to come down from the district office to grab him. Vasya is a respected person. He's a thief."
"And me?"
"You're an enemy of the state. If the neighborhood cops can decide to arrest Vasya, the Secret Chamber, so to speak, would have to give the orders to arrest you. The decision to arrest you would be made by no one lower ranked than Bortnikov's deputy, although you're naked and barefoot, and no one would ask the prosecutor's office or the Investigative Committee to go after you. It's creepy and pointless."
This should give you an idea of the conversations I have with my acquaintances in the security forces nowadays. It helps to do business with people who know the score. None of them is surprised when you ask them who would arrest someone, how they would do it, and when they would do it. Everything would have been planned long ago, and there are no illusions. If a person has to be placed under arrest and charged, it is going to happen. If they do not need to be indicted, they can be kept in custody for a while. No one remembers, even for appearance's sake, that there are courts in Russia, and courts decide whether to remand someone in custody after hearing arguments by all the interested parties. Everyone knows the decisions are not made in court.
Who Makes the Decisions?
Who made the decision to arrest Kirill Serebrennikov? Who decided to let him go for the time being? Who arrested Michael Calvey and the employees of Baring Vostok? Who let them out of jail? Why? Who made the decision to arrest Mikhail Abyzov?
There is no one with whom you can talk about these cases.
This is not quite true. My sources in all the law enforcement and security agencies, who can be frank with me as long as they remain anonymous, talk to me about these cases, too, but they look really worried when they do.
Rank-and-file law enforcement officers are confused. They do not understand why someone decided to back off the Serebrennikov case so abruptly and quickly. The train was rushing the director and filmmaker towards a sentence of the four years or so in the camps when a powerful hand jerked hard on the brakes. The passengers jumped off the train, of course, for they didn't want to keep traveling in that direction, but the trainmaster, driver, and conductors were completely at a loss.
What should they do with the next train and its contingent of VIP passengers? Should they railroad them, as they were ordered to do, or should they avoid hurrying the case? After the emergency brake has been pulled, everyone emerges with injuries and bumps. Some of the crew were counting on promotions after they had wrapped up such a big case. Other members of the crew were acting on orders from a celestial. He will not forgive them because now they know there are tougher celestials in the system. He cannot forgive the people involved in the case for knowing that fact nor can he forgive the other celestials for intervening. The passengers could not care less. Either they get to where they are going or they do not get there, but the crew is always aboard the train.
True, a smart alec from the Investigative Committee told me something interesting about the procedural aspect.
"Why is everyone so angry? The Serebrennikov case was sent back to the prosecutor's office, so what? You saw that the court ordered a forensic examination. The first forensic examination was really crooked. The judge in the trial of Serebrennikov's accountant, Nina Maslyaeva, wondered why everyone was so glad. Serebrennikov's case would now be sent back to the prosecutor's office because his circumstances are the same as Maslyaeva's. You are mixing up cause and effect. The judge in the Maslyaeva case cannot reach a verdict because he understands the outcome of the forensic examination, which was the same as in the Serebrennikov case, will now be different, and Maslayeva will have to be re-indicted in the light of the new forensic examination in the Serebrennikov case."
Translated into ordinary language, he means the case can still go any which way. Procedurally, all the cards are still on the table, and the haggling could continue. Things could go one way or the other. The powers that be could change their minds and send Serebrennikov to prison, but they could also let him go. They could arrest him again and send him down. The statute of limitations is a flexible thing.
Somewhere above the clouds, the thunder gods fight over the case. Invisible to the world, they communicate with ordinary people by making motions to conduct additional forensic examinations. Ordinary people make of it what they will. Police investigators are also part of the rank and file, part and parcel of Russia's unwashed masses.
In ordinary times, this is not what happens to ordinary defendants in ordinary cases. Everyone would have gone down five years each per capita, and no would have batted an eye. In this case, the decisions are obviously political. Look who made the decision! Who telephoned whom? What levers did they use? Who or what did they offer in exchange? Freebies are for freaks, after all. We will return to this subsequently when we discuss other factors.
If the boring procedural hypothesis made by my anonymous source at the Investigative Committee is right, events should unfold as follows. The authorities will get the results of the new forensic examination in the Serebrennikov case. If the total damages are less than was claimed earlier (or, say, there were, miraculously, no damages at all), the charges against Serebrennikov and the other defendants will be dropped right in the courtroom. If, on the contrary, the sum of the damages is more or less hefty, a million rubles, at least, the defendants will be found guilty and sentenced to prison. Then you can appeal the verdict wherever you like.
No one would ask why a particular ruling was made. No one would ask what happened. Why are some people treated one way, while others are treated another way? The foot soldiers of law enforcement know the score. But when they do not know the score, they know it is better not to ask whether a mistake has been made but to follow orders.
How Things Go Down
The Calvey case bears a strong resemblance to the case against Vladimir Yevtushenkov. Yevtushenkov failed to take the hints. He was told directly what to do but refused to hand over his business. Then he was arrested and given a good talking. He and his captors came to an understanding. He was released and his business confiscated. Unlike Yevtushenkov, however, Calvey is as poor as a church mouse. Compared with Yevtushenkov, that is. Calvey does not own a Bashneft, after all.
The foot soldiers in the security forces have not been particularly surprised about how the Calvey case has unfolded. They expected something of the sort. They expected him to "cash out," as they call it, and they believe he has, in fact, cashed out. They are uninterested in what this meant. It is not their war, and the spoils are not theirs to claim.
We should look at this more closely.
My source, whom I trust, albeit warily, explains the obvious to me.
"All cases are business as usual except the cases in which there a phone call," he says.
I have two questions for him right off the bat. What does he mean by "business as usual"? Who usually makes the "phone call"?
He explains that people who follow high-profile cases and comment on them fail to take one important factor into account in their arguments. The high-profile cases are handled by another agency as it were. They involve the same players: the prosecutor's offices, the courts, the remand prisons, and the Investigative Committee. All of them realize, however, when they are handling a special case involving the interests of high-ranking officials and elite businessmen. In these cases, they need to keep close track of which way the wind blows.
The bulk of cases are "mundane." There is a huge number of such cases, and they can drag on forever. Take, for example, the Baltstroy case, the case of police anti-corruption investigator Boris Kolesnikov, and the case of ex-deputy culture minister Grigory Pirumov, cases that everyone has forgotten, and the Oboronservis case, the cases of the banks implicated in the so-called Russian Laundromat, and the case of Alexander Grigoriev, the man, allegedly, behind the Laundromat, who was mixed up with Putin's cousin Igor Putin. New indictments in these cases are made all the time. More and more defendants are convicted in these cases and sent down. It never stops, but public interest in these cases is almost nil.
There are cases that collapse, however. Why does this happen?
Why was the case of ex-economics minister Alexei Ulyukayev not reviewed on appeal? Why was his prison sentence not reduced by four years during the sentencing appeal hearing? Does anyone know why? Perhaps the political spin doctors get it, but Russia's law enforcers do not have a clue. What they understand is when an order comes down to reduce a sentence and when it does not. They leave the blabbing to the spin doctors.
Alexei Fedyarov is a former prosecutor from Chuvashia. Nowadays, he is the head of our legal department at Russia Behind Bars. He gave me permission to quote him.
"It happens. A case is going fine. In the morning, you have a meeting with your superiors. They tell you everything is great, keep pushing, you've got the bastards. I was handling a case against the management of the Khimprom factory in Novocheboksarsk. At briefings, I was told my group and I were doing a great job. We had done the initial investigation beautifully and now it was time to detain the suspects, remand them in custody, and put them away. I went to my office, where the city prosecutor was waiting for me. He asked me to hand over the case file. I gave him the case file and he told me it was over, I should forget it. He was personally going to deliver the case file to the head prosecutor of the republic and that would be the end of it. There would be no supporting documentation or anything. The case really did disappear, although an hour before I had been told to push it.
"During that hour, the head prosecutor of the republic had got a message from the Russian Prosecutor General's Office. A call from a deputy prosecutor general was enough for them to take the whole thing back, despite the fact it was a big, interesting case involving illegal wiretapping throughout the company and even the local police department and the tax police office. We had found tons of recorded conversations: they recorded everything. They were trying to protect themselves and investigate other people."
Sources of the "Telephone Call"
How does the "telephone call" work?
The "telephone call" is a conventional name for the outcome of lengthy negotiations. We see only the reflection of this process: Calvey's arrest, his transfer to house arrest, Serebrennikov's arrest and his release on his own recognizance, Abyzov's arrest.
I am going to quote my anonymous source verbatim. In this instance, the way he says what he says is as important as what he says.
"Anyone can hit the brakes. It could be Bortnikov. It could be Chaika. But it is the outcome of agreements among people, not an arbitrary decision. They do not do things that way. Maybe new factors have been brought into play, but there has to be someone who wants to negotiate on behalf of the accused person, who appeals on his behalf. He would be told, 'Okay, fine. But you have to give us such-and-such in exchange." Then it is a matter of talking with Lebedev [Chief Justice of the Russian Supreme Court] and everything is put into reverse. It could be like, 'We'll give up Serebrennikov if you take the heat off Calvey.' You see, the siloviki are not all on the same side. There is no longer one side. Not even everyone in the FSB or its departments is on the same side. The Constitutional Department fights with the Anti-Terrorism Department. It's the same thing in the prosecutor's office and the Investigative Committee. In the Investigative Committee, there is the group loyal to Bastrykin and then they are the boys from the North Caucasus. There are also the guys from Tatarstan and Bashkortostan, who are filthy rich but live orderly lives and are also capable of getting things done.
"Anything goes at this level. Why are you inclined to exaggerate how this works? Number One basically does not care about this stuff."
I should try and explain.
The Investigative Committee and Prosecutor General's Office are still at serious loggerheads. The conflict has even intensified. It is a personal conflict and a clash of business interests and a fight over resources. The amount of resources has not grown. On the contrary, there are palpably fewer resources. Relations between the Investigative Committee and the Prosecutor General's Office are currently not just strained, they are intolerably strained.
In court, they take the same side, but those are the rules of the game. If a case has gone to trial, you cannot come out against your colleagues: you would be digging yourself a hole. As a prosecutor, you did not reverse the indictment. You were involved in prolonging the suspect's custody in remand prison, and you seconded all the motions made by the case investigator. The case investigator, of course, always plays along with the prosecutor. In criminal trials, they are the prosecution.
Even the "groundlings" find it easier to make a deal. The big bosses may be at war with each other, but down on the ground, the workhorses plow away and know the score. There is no love lost for Bastrykin among Investigative Committee officers just as prosecutors are not fond of Chaika. But it is like this everywhere: people like their bosses only when they are standing right in front of them. There is a certain difference, however. Chaika and his deputies at the Prosecutor General's Office are all former case investigators. They have paid their dues. Bastrykin does not have this background: he is not a criminologist. Their workhorses thus complain about different things. Bastrykin's underlings complain about incompetence, while prosecutors grouse about their bosses' passion for business.
The Investigative Committee and the Prosecutor's Office have an innate tendency to divide up into clans, which are defined geographically: there are Circassian clans, Bashkir clans, etc. They are local fraternities of sorts, and they do not go away when someone moves and transfers to a new job. The clans are often at odds with each other. This is something you must always factor in when dealing with Russian law enforcers.
Internal disunity has also been increasing day by day in the conglomerate known as the FSB. Even mid-level officers have trouble getting along. For example, M Directorate, which oversees the Interior Ministry, the Federal Penitentiary Service, and so on, is often combined, in many regions, with the Economic Security Department, and there is a big problem with compatibility in terms of the cases they pursue. But there is also K Directorate, aka the 8th Directorate, which oversees banks and the financial system. Regarded as "blue bloods," they are strongly disliked by other FSB officers.
"A guy from K Directorate worked out at the World Class gym where I worked out. His driver took him to work in a Maybach. Now he has transferred his membership to the gym in Zhukovka. A membership there costs 600,000 rubles a year [approx. $9,500] and the swimming pool is filled with mineral water. 'My clients work out there,' he said to me, 'so I moved my membership there,'" an athlete and retired FSB veteran told me.
The FSB's Constitutional and Anti-Terrorism Departments are a whole other story. They oversee everyone who has any dealings with the opposition and they inspire no confidence whatsoever. For example, I am flattered Kirill Serebrennikov and I are overseen by the same FSB officers. But we are overseen by officers from the Constitutional Department, while the Anti-Terrorism Department are working-class blokes who specialize in completely different cases. They were merged into a single directorate in which the Anti-Terrorism Department, supposedly, is subordinated to the Constitutional Department. Naturally, they cannot stand each other.
What about the top bosses? They are busy with other things, which is why they are in charge. They are busy with politicking and intrigues. These quiet squabbles surface as cases like the recent arrest of Colonel Kirill Cherkalin from K Directorate. Did he really take a bribe? Maybe he did: anything is possible. It is more likely, however, he was arrested as part of a war for turf, turf that has been shrinking exponentially with every passing day. Fattened cows no longer graze on this turf: there are basically no cows left to milk. The entire herd has been devoured.
I will quote in full the monologue my anonymous source delivered when I asked him about the future. I do no think there is any need to decode it.
"The turbulence will increase. Until all the issues with Russia's natural gas and its transit through Ukraine are settled, Number One won't have time for things happening here. They have been outsourced to our guys. They have been told to go and bite everyone's heads off. They have temporary permission to do it.
"But there are few fat cats. All the money has been sent abroad. Everyone is living on loans. All of Rublyovka is up to their ears in loans. There will be searches in some people's homes, and some folks will be ripped to shreds. There will be a lot of this kind of stuff this year. The government will be purged, too. People love this sort of thing.
"Abyzov made no impression on anyone. No one understood what it was about. The only thing people will remember is that he offered to pay a billion rubles in bail. No one will forget him and the billion rubles.
"Circumstances are such that even the system's insiders cannot make any forecasts. The settings are changing constantly. There is no stable paradigm.
"It is like with water. At room temperature, we understand how it acts. You can stick your finger in it and blow on it. But now it is being warmed. It has not boiled yet and vaporized, but you do not know what to do with it and how it will act next.
"The tax police are busy with major shakedowns. They are kicking everyone's ass. When we ask them why they are doing it, they reply, 'Crimea is ours, and our job is to get people to make additional payments.' But additional payments and penalties are different things, especially penalties meant to wipe people out. They are going after people's last rubles.
"I have a friend who works as a business court judge on tax cases. Whereas earlier, when she would be asked why she reduced a claim from one hundred million rubles to ten million, she could have an off-the-record chat with the head judge of the court and explain she was doing it so the person could keep their business, such chats are not kosher nowadays.
"Hard times are coming. The Syrian project fell through, and Russia failed to get control of the pipeline going through Turkey. Nothing that was planned in Syria has worked out, and both the South Stream and Nord Stream projects fell through [sic]. Nor will they replace the Ukrainian transit, although that was the goal. But it impossible to exit Syria, and now they have butted their noses in Venezuela. Their luck has been bad. People's nerves are on edge up top.
"Number One is interested only in oil and gas, and so other parties have got involved in the game. If it were up to Number One, he would crush everyone and no one would breathe another word. He probably decided the lower ranks should take care of this stuff themselves. The very top bosses are not concerned with these matters at all right now. The lower ranks are running things and a huge amount of haggling has been happening. We are witnessing a classic turf war."
Welcome to the magical world of turbulence in a pot of boiling water.
Olga Romanova is the director of Russia Behind Bars, a charitable foundation that aids Russian convicts and their families, people who have been victimized by the Russian justice system. Translated by the Russian Reader
By hecksinductionhourin economy, putinism, regime, zeitgeist July 22, 2019 July 22, 2019 3,550 WordsLeave a comment
Vladislav Inozemtsev: The Calm before the Storm
A common sight: first-floor commercial space for rent in downtown Petersburg.
The Calm before the Storm: Can We Avoid Economic Collapse in 2018?
Vladislav Inozemtsev
Last week, Tatyana Nesterenko, one of Russia's most experienced financiers and a person distant from politics, a person who has held the post of deputy finance minister and head of the Federal Treasury for almost twenty years, said the Russian economy should expect serious financial problems as early as next year, comparing the current situation with the "eye of a storm, [meaning] a condition in which everything [merely] looks quiet and safe."
In my view, Nesterenko is undoubtedly right. The government has recently appeared to be the epitome of tranquility. It has even been drafting a new three-year budget, although in terms of revenues for 2016, the previous such plan (for 2014–2016) was off by 42%! Revenues were projected at 15.9 trillion rubles, but actual revenues in the first six months of the year were 4.6 trillion rubles. I don't think the new draft budget will prove more accurate, if only because no sources of income for covering the deficit are envisioned after 2017. The president, who from time to time meets with economists and recommends developing a new development strategy for the country "roughly within a year," meaning when the Finance Ministry's reserves will run out and the budget's huge social commitments will prove impracticable, has mainly been busy reshuffling the security forces, believing, apparently, that a sum changes by rearranging its components.
Coins tossed for good luck onto a cable spool anchored in the Fontanka River.
Today, Russia's economy, to invoke Economic Development Minister Alexei Ulyukayev's maxim, really has hit rock bottom. The authorities are elated that the rate at which the GDP has been falling fell to 0.6% in the second quarter, but we should note this reduction took place in conjunction with an accelerating reduction of real incomes (by 6.2% in May, and 4.8% in June) and a considerable increase in inflationary expectations. Annual inflation was 7.5% as of June and showed no tendency toward decreasing.
Moreover, oil prices have fallen considerably. Brent fell by 15.2% during July, and, apparently, black gold is near a new equilibrium price ranging between 38.5 and 43 dollars a barrel. A 15–16% fall in the oil price will cost the Russian budget 430 to 460 billion rubles in the remaining five months of 2016, which is also no cause for optimism. Responding to it by "managing" the descent of the ruble will not be easy. Devaluing the national currency will no longer lead to a growth in exports, which this year has lagged behind last year's figures by 30.5%. On the other hand, imports, which have basically not shrunk (they are down by only 10.4%) will inevitably become more expensive, dragging along with them the prices for a wide range of goods, thereby causing inflation and setting the tone for high interest rates.
Empty billboards are also not hard to come by in the city center.
In mid 2016, the Russian economy really is situated in the eye of a kind of storm. It is quite calm there at the moment: the authorities have become accustomed to the new circumstances. They have no hesitation in spending reserve funds. Generally, fears of popular discontent over lowing living standards have been overcome. Seemingly, a certain reduction in the degree of hostility toward western countries might do the trick of restoring relations with them. There has been a glimmer of hope the EU's problems will deepen with the UK's exit. The possibility of Donald Trump winning the presidential race in the US has been taken seriously. Putin feels like a winner in his confrontation with Turkey. It is no wonder officials have dubbed the situation the "new normal." It really is the new normal, so as long we take into account two factors: oil at 50 dollars a barrel and spending accumulated reserves at the rate of 600 billion rubles a quarter. That is around 8% of the overall amount in both sovereign wealth funds, the Reserve Fund and the National Wealth Fund.
Another empty billboard
However, the problem is not so much that sooner or later we will have to break back into the open sea through the hurricane's eye wall, but the fact that the eye of the storm might move, and it would appear we have no instruments for tracking it. The country has not been trying to find the best place in this "quiet corner." It has simply been drifting, humbling waiting for what happens next.
Evaluating the numerous programs and strategies that experts affiliated with one or another wing of the government are now trying to draft, one cannot help thinking that none of them is capable of boosting the Russian economy in view of two circumstances.
An elderly woman turning in scrap paper and other junk to supplement her pension.
On the one hand, anti-crisis measures should have been implemented yesterday, rather than postponing their preliminary discussion to 2017. By the way, Russian Finance Minister Anton Siluanov warned in early 2016 that, at current oil prices, the Russian budget would be short 3 trillion rubles, which in turn would lead to spending the greater part of the National Wealth Fund. Little has changed since then. In the absence of reserve funds, the hole in the budget cannot be closed in 2018 either by raising funds on the international capital market (in this case, we would have to raise considerably more money than all the central government's current international obligations) or by privatizing. (One year's deficit could be papered over only by selling off the lion's share of the state's holdings in Gazprom and Rosneft.) Whatever economic development strategy the Kremlin approves a year from now, it will not prevent a large-scale collapse in 2018, with all the attendant consequences.
On the other hand, all the existing programs, however much Alexei Kudrin and Boris Titov stress their differences, are generally focused on the same thing: relaunching the economy on behalf of manufacturers. There is in fact only one difference between them. Titov's Stolypin Club has suggested priming major enterprises with money through the earmarked and regulated distribution of cheap loans, subsidized by the Central Bank, while Kudrin's Center for Strategic Research favors institutional reforms that could include reducing taxes on business and limiting the rights and opportunities of security services and the bureaucracy for extracting additional income from business. The assumption is that either by getting its hands on cheap money or ridding itself of the unbearable pressure of regulators, business will be reanimated, sparking life-saving economic growth.
More commercial real estate for rent in the city center.
I would love to be wrong, but I don't think these measures will produce any meaningful outcomes, because the most important factor in the economic slowdown of 2014–2016 has been the crisis in consumer demand. The state has diligently performed its investment obligations, saturating heavy industry with funds via defense sector orders. It has not halted its sometimes pointless but expensive infrastructure projects. It has been encouraging state companies to build new pipelines and railways, but none of it can compensate the effect of declining consumer demand. Moreover, this demand has increasingly shifted towards the continuing flow of imports, while the share of domestic goods on the market has stopped growing. As I understand it, none of the economic development programs has so far offered a solution to this problem.
Therefore, in my opinion, we should introduce as least three new story lines into the ongoing debates.
First, we should stop regarding increases in wages for low-paid earners, pensions, allowances, and other payments to low-income Russians as "costly measures." Saving money by reducing the incomes of doctors, teachers or pensioners is much more destructive than reducing costs at Gazprom or expenditures in the program for rearming the Russian army. These segments of the populace are most focused on purchasing domestic goods and services, and investing in them produces a multiplier effect in the sales and production of consumer goods. The crisis of 2008–2009 was negotiated much more successfully than the current crisis not only due to the relatively radical reversal in oil prices but also because the government considerably increased people's incomes at the time, despite budget problems, whereas 41% of the population now say they lack money for food and clothing.
Second, we should think hard about a one-time credit and debt amnesty for people whose indebtedness to banks, the tax authorities or housing and utilities sector companies does not exceed, say, 30 thousand rubles. Obligations of this amount now account for around 20% of the population's entire debt burden, and a measure like this would affect 10 to 12 million people. The state would have to allocate up to 2 trillion rubles to implement the program, but both the social and political (why deny it) effect of such a measure would be incomparable with a Stolypin Club-style emission of a similar scale, which would completely vanish in the offshore accounts of executives of major companies in bed with the state and sympathetic officials. In my view, we cannot do without this measure now, but none of the people involved in the current discussion has deigned to mention it in their programs.
Third, direct measures for stimulating demand are necessary. They were adopted by all the governments of developed countries hit by the crisis of 2008–2009, but our officials were quite reluctant to copy them. I have in mind not only programs for encouraging purchases of new automobiles but also a system of issuing food stamps, analogous to the American one, to poor people. For example, pensioners could buy stamps nominally worth four to six thousand rubles for two to three thousand rubles at welfare offices. The stamps would be accepted in shops as payment for domestic food products only, with the exception of cigarettes and alcohol, and commercial outlets would then turn them in at banks at face value and have the amount credited to their accounts. This could be a powerful stimulus both to domestic manufacturers and commerce, not to mention the popularity of such a step among socially vulnerable groups themselves.
Ads like this one for a prostitute service are stenciled and pasted on every available surface in the city center.
In other words, now it is not enough to say that Russia has sailed into a perfect storm. It must be understand that not only the captain and his mates will have to fight for our ship's survival but absolutely all the passengers as well, and so the basis of an anti-crisis program should be attention to the general population, not to state corporations. And, of course, to be at least relatively prepared to fend off the mighty blows of the elements, we must stop postponing actual steps until tomorrow, and begin taking them today.
Source of original text: Worldcrisis.ru. Translated by the Russian Reader.
By hecksinductionhourin capitalism, economy, politics, putinism, society August 8, 2016 1,840 Words1 Comment


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text:
German machine tool makers close 2021 with full order books
Orders received by the German machine tool industry in the fourth quarter of 2021 were 51 percent up on the previous year's figure. Orders from Germany rose by 62 percent. Foreign orders were 46 percent higher than in the previous year. In 2021 as a whole, the level of orders received by German manufacturers rose by 58 percent. Domestic orders were 51 percent higher than in the previous year, foreign orders 62 per-cent.
"This great result reveals how German manufacturers have worked their way out of the Covid crisis," says Dr Wilfried Schäfer, Executive Director of the VDW (German Machine Tool Builders' Association), Frankfurt am Main, commenting on the result. "Orders are also 11 percent higher than in 2019, the year before Covid," Schäfer adds.
The recovery in foreign order levels is broad-based across all the different regions. Europe recorded the strongest growth at 90 percent. The Americas and Asia were up 66 and 61 percent, respectively, on the previous year. Within Europe, both Italy and Austria were supported by massive investment funding programs and came back very strongly, as did the Czech Republic. While the level of orders from Austria only returned to normal in the second half of the year, that from Italy remained at a high level throughout the year.
The two lead markets, China and the USA, also performed strongly. Overall, however, the USA has shown stronger momentum and narrowed the gap to China.
"All in all, there are great prospects of the sector having a good year in 2022," Schäfer concludes. "We are expecting the wave of Covid-19 infections to weaken in the coming weeks and many restrictions to fall away. Most manufacturers also expect supply problems to ease in the second half of the year.
In particular, the bottlenecks in the logistics chains should resolve themselves,allowing companies to process their orders and deliver their machines more smoothly."
German machine tool makers close 2021 with full order books (pdf) (pdf)

redpajama_set_name: RedPajamaCommonCrawl


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Research ArticleMOLECULAR EVOLUTION AND GENOMICS
Comparative Sequence Analysis of Colinear Barley and Rice Bacterial Artificial Chromosomes
Jorge Dubcovsky, Wusirika Ramakrishna, Phillip J. SanMiguel, Carlos S. Busso, Liuling Yan, Bryan A. Shiloff, Jeffrey L. Bennetzen
Jorge Dubcovsky
Wusirika Ramakrishna
Phillip J. SanMiguel
Carlos S. Busso
Liuling Yan
Bryan A. Shiloff
Jeffrey L. Bennetzen
Published March 2001. DOI: https://doi.org/10.1104/pp.125.3.1342
Copyright © 2001 American Society of Plant Physiologists
Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3′ untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.
The grass family is the fourth largest family of flowering plants (approximately 10,000 species) and includes wheat (Triticum spp.), maize (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), and oats (Avena sativa). This diverse family exhibits a large variation in DNA content. Rice is a diploid species with a relatively small genome (430 Mb), whereas barley and many other diploid Triticeae spp. contain 4,000 to 8,000 Mb of DNA in their nuclei (Bennett and Leitch, 1995). These differences in DNA content indicate that a single chromosome from barley contains more DNA than one complete haploid rice genome.
Despite the enormous differences in genome size, comparative RFLP maps have revealed a remarkable conservation of gene content and gene order among the cereal genomes (Gale and Devos, 1998). Early DNA renaturation studies had indicated that the larger plant genomes contain more repetitive DNA and that some of this repetitive DNA was intermixed with genes (Flavell and Smith, 1976). The sequencing of a 280-kb segment around the adh1-F gene in maize, a large genome grass (2,400 Mb), showed that intergenic regions were mainly composed of retrotransposons inserted within each other. These retro-elements accounted for more than 60% of the DNA in the adh1-F interval and more than 70% of the total maize genome (SanMiguel et al., 1996; SanMiguel and Bennetzen, 1998). Comparison of the orthologous region in a close relative with a smaller genome, sorghum (Sorghum bicolor; 750 Mb), showed a complete absence of detected retro-elements. Only genes identified within this interval were evolutionarily conserved, indicating an extensive differentiation in the intergenic regions of these species (Chen et al., 1997).
The diploid Triticeae genomes are even larger than the maize genome (barley = 5,000 Mb; rye [Secale cereale] = 8,000 Mb) and repetitive sequences represent more than 75% of the genome (Flavell and Smith, 1976; Suoniemi et al., 1996; Gribbon et al., 1999;Vicient et al., 1999). Panstruga et al. (1998) sequenced a 60-kb region in barley around the mlo locus and found three genes plus retrotransposons and complex repeat structures that accounted for one-quarter of the DNA in this interval. Similar repeat structures accounted for more than one-half of the DNA sequence in another 66-kb region in barley around the rar1 locus (Shirasu et al., 2000). Even though these and other preliminary sequencing efforts in Triticeae species (Wei et al., 1999; Stein et al., 2000; Lagudah et al., 2001) are providing a first picture of the organization of these large genomes, no large segment of Triticeae DNA sequence has been compared yet with its orthologous region in rice. Preliminary comparisons of this sort will help indicate to what degree the large amount of rice sequencing generated by the Rice Genome Project will be useful for positional cloning and genetic characterization in the Triticeae.
This paper presents the DNA sequence and sequence comparison of an orthologous region of the rice and barley genomes. Our data show that comparative sequence analyses of this type can greatly assist sequence annotation, including gene identification and characterization of gene structure. More importantly, these studies will help indicate the nature, frequency, and timing of different types of sequence evolution in these two important grass lineages.
Clone Identification
Thirty-seven thousand and 300,000 clones from rice and barley bacterial artificial chromosome (BAC) libraries, respectively, were screened for individual clones that contained homology to WG644. Four clones with homology were identified in each library and clones 635P2 (barley) and 36I5 (rice) were selected for sequencing because they appeared to have the greatest degree of overlap. Both BACs hybridized also with rice RFLP clones R2311 (two copies in 36I5) and R2628 and barley subclone UCW12 from the plasmid rescue of BAC 635P2. Barley subclone UCW12 was positioned on a Triticum monococcum genetic map (Dubcovsky et al., 1996) and found to be completely linked to WG644. RFLP markers R2311 and R2628 were also found to be closely linked to WG644 in different rice genetic maps (Maroof et al., 1996; Harushima et al., 1998; Sarma et al., 1998; Kato et al., 1999).
BAC Sequencing
Rice BAC 36I5 was sequenced at different levels of coverage (5×, 10×, 15×, and 20×; PHRED > 20) and assembled after each round of sequence to evaluate the best balance between initial coverage and final finishing efforts. The number of gaps and "problems," defined as areas covered by a single subclone or areas with a base quality below 1 in 10,000 error rate, decreased with the increasing coverage (5×, six gaps and 216 problems; 10×, three gaps and 80 problems; 15×, two gaps and 43 problems; and 20×, two gaps and 39 problems). Although the gap sizes were reduced, other problems were not efficiently solved by increasing coverage from 15× to 20×. One problem region on this BAC, containing two duplicated regions in inverted orientation from the end of gene 4, adjacent to a tandem of five large repeats, was not fully resolved, resulting in two contigs of 66,700 and 5,812 bp. We report here the sequence of the first 50,000 bp with a predicted error rate lower than 1 in 100,000 bp (GenBank accession no. AY013245).
Barley BAC 635P2 was completely assembled, starting from a 13× redundant set of shotgun sequences (PHRED scores > 20). Difficult regions such as G- and C-rich areas and gaps in the region of two nested BARE-1 retrotransposons in barley BAC 635P2 were closed using a combination of primer walking and drhodamine and dGTP big dye terminator chemistries. The complete sequence was 102,433 bp and has a predicted error rate lower than 1 in 100,000 bp (GenBank accession no.AY013246).
Colinear Gene Region
The first step in the sequence analysis of these two colinear BACs was the delimitation of regions that are conserved and unique between the rice and barley sequences. Unique regions were annotated individually for each BAC, but conserved regions were annotated simultaneously in both BACs and compared with the information provided by the rice-barley sequence alignment and six-frame translation of the homologous regions.
Comparison of the complete sequence of 36I5 with the complete sequence of 635P2 using the program Dotter revealed the presence of four conserved regions (Fig. 1). The same regions were detected by the gene prediction programs Genescan and GeneMark.hmm and by BLAST and GeneSeqer searches of the expressed sequence tag (EST) databases, suggesting that they correspond to gene regions. No similarity was found between the rice and barley sequence outside the genes with the exception of short stretches of homology in the promoters and 3′-untranslated regions and non-colinear Stowaway transposable elements.
Sequence comparison of barley BAC 635P2 with the first 50 kb of rice BAC 36I5 using the program Dotter (word 25, similarity 80). The location of genes and large transposable elements in rice (vertical) and barley (horizontal) are indicated by arrows and boxes, respectively. A more detailed annotation is presented in Figure2.
The conserved regions span 30-kb at the 5′ end of the rice sequence and the complete 102 kb of the barley sequence. The greater physical length of the four-gene region on the barley BAC was due to larger intergenic spaces than in rice. Distances between genes 1 and 2 (rice = 1.4 kb; barley = 24.0 kb), and between genes 2 and 3 (rice = 0.6 kb; barley = 30.5 kb) were greatly expanded in barley by the insertion of different transposable elements (see below). Gene 4 was duplicated in barley (gene 4a and 4b) but not in rice (Figs. 1 and2).
Organization of repetitive and conserved sequences in barley BAC 635P2 and the first 50 kb of rice BAC36I5. Genes are represented by arrows, transposable elements by boxes, and MITEs by triangles. Ta and Tb,Copia-like retrotransposon; Tc, non-LTR retroelement; Td, terminal-inverted repeat element;Te and Tf, nested BARE-1 retrotransposons;Tg, MuDR-like element; Th,Copia-like retrotransposon; Ti, nonautonomousMutator element. SSRs with eight or more repeats are indicated by an asterisk.
The four genes are in the same predicted transcriptional orientation in rice, but the second gene is in inverted orientation in barley (Figs. 1and 2). The relative inversion of gene 2 was confirmed experimentally. All fragments observed on a pulse field agarose gel with digestions of both BACs with eight-cutter restriction enzymes and all possible double digestions agreed with the restriction maps predicted by the current sequence assembly (Fig. 2). An additional gel replica was prepared with DNA from both BACs digested with eight six-cutter restriction enzymes, several of them with restriction sites between exons 3 and 9 from gene 2. Filter replicas from the six- and eight-cutter restriction enzymes were hybridized with two probes from genes 2 and 1 probe from gene 1 to analyze putative short inversions. The first probe for gene 2 included exons 1 to 3 and the second one exons 9 to 13. Restriction fragments detected by hybridization with the three probes were all predicted by the current sequence assembly, suggesting that the relative inversion of gene 2 is not the product of an incorrect sequence assembly.
Two areas of inverted homology were identified flanking barley gene 2 (6,893–7,457 and 38,348–35,605 bp; Fig. 2). These inverted repeats might have been associated with a recombination event between nonorthologous repeated sequences located in opposite orientation at both sides of gene 2, resulting in the inversion of this barley gene.
The four Arabidopsis genes with the highest similarity with the four genes identified within the rice and barley BACs (TableI) seemed to be not colinear in Arabidopsis. The closest Arabidopsis homologues for rice and barley genes 1, 2, and 4 (Table I) are all located on Arabidopsis chromosome 5, but on three different BACs positioned approximately 10,000 kb from each other on the physical map (BAC F14F8 = 5,400–5,500 kb; MSL3 = 23,700–23,800; MXF12 = 14,400–14,500). The closest homolog for barley and rice gene 3 maps to Arabidopsis chromosome 1 (BAC F4N2 = 25,100–25,200 kb).
Table I.
BLASTP comparisons between the barley predicted protein, the rice predicted protein, and the two most similar Arabidopsis proteins
An additional search of the Arabidopsis database was performed using TBLASTN and the four Arabidopsis predicted proteins concatenated in a single file. The objective of this search was to explore the possibility of genes, which are colinear between the grasses and Arabidopsis but which have diverged considerably in sequence. This search showed that the Arabidopsis gene MXF12.5 adjacent to the closest Arabidopsis homologue for barley gene 4 (MXF12.6, Table I) was similar to barley gene 3, although with BLAST scores (score = 70) one order of magnitude lower than the S1K1 closest homolog (score = 797). Significant BLAST scores were also obtained between two grass genes and the Arabidopsis BAC MSL3. However, in this case the closest homolog of gene 2 (MSL3.6) was more than 20 kb apart from the Arabidopsis gene showing similarity with barley gene 3 and that similarity was very low (score 67.6 Expect 5e-10). These weak similarities to gene 3 in the proximity of the closest Arabidopsis homologues to genes 2 and 4 are more likely related to the abundance of kinase genes similar to S1K1 than to true conserved pairs of colinear genes.
The Nonoverlapping Region from Rice BAC 36I5
Gene predictions in the 20-kb of the BAC 36I5 contiguous sequence that does not overlap with BAC 635P2 were more difficult because of the absence of comparable barley sequence. However, both Genescan and GeneSeqer predicted an initiation codon at 39,753 bp and a terminal exon at 40,748 bp tentatively designated as gene 5. The presence of a gene in this region was further supported by the significant similarity between three nonoverlapping segments from this gene with three rice cDNA clones (C27091 from 11–280 bp, 95% identical; C74904 from 320–519 bp, 92% identical; and C27330 from 707–883 bp, 95% identical) (Table I). Alignment with C74904 suggested different exon-intron boundaries to those predicted by Genescan. Assembly of a protein from the putative coding regions was performed and analysis of the putative protein by BLASTP and TBLASTN produced no matches to known proteins.
The comparison of the colinear barley and rice sequence not only delimited the genes within the genomic sequence but also helped to establish their structure.
The predicted protein from barley gene 1 has a high level of similarity with the predicted rice protein (93% similarity, Table I) and with the closest Arabidopsis homologue (gene F14F8.20, 353 aa, 73% similarity). All three genes are divided in six exons (Fig.3). Exon 2 has identical size in all three species and exons 3 to 6 have identical length in barley and rice. However, analysis of the Arabidopsis genomic sequence revealed the existence of alternative GT (19 bp downstream from the current annotation) and AG (71 bp upstream from the current annotation) splice sites between exons 3 and 4. If these alternative splice sites are used, the third exon of the predicted Arabidopsis protein would have the same length as the rice and barley exon 3 and the overall similarity between Arabidopsis and barley predicted proteins would improve from 73% to 81%. This alternative annotation is presented in Figure 3. Gene structure in barley was confirmed by the absence of gaps in the alignment of the DNA sequence from the predicted barley cDNA (concatenated predicted exons) with a cDNA from Triticum durum (BE429317, 97% identical) and a cDNA from cold stressed winter rye (BE704834, 96% identical, Table I). The closest Arabidopsis homologue has been annotated as a putative mitochondrial carrier protein by the presence of a mitochondrial energy transfer protein signature (CAC01763).
Structure of the genes found in the regions conserved between rice BAC 36I5 and barley BAC 635P2 and the closest Arabidopsis homologues (Table I). Exons (boxes) and introns (lines) are proportional to their length. Exons of identical length in the three species are indicated in black and those of identical length in rice and barley in black with white diagonal lines.
The predicted protein from barley gene 2 also has a high level of similarity with the predicted rice protein (88% similarity, Table I) and with the closest Arabidopsis homologue (gene MSL3.6, 425 aa, 72% similarity, Table I). The rice and barley genes are divided in 13 exons, 10 of which are of identical length (Fig. 3). The Arabidopsis gene has seven exons of identical length to the grass species but has one exon less. The last exon of the Arabidopsis gene showed 79% similarity to the last two exons from rice and its length (210 bp) was identical to the sum of the lengths of the last two exons in rice (57 and 153 bp). Analysis of the Arabidopsis genomic sequence showed the presence of alternative splice sites that would improve the similarity between the Arabidopsis predicted protein and the predicted proteins from grasses. An alternative GT splice site 87 bp downstream of the annotated end of exon 1 would determine an exon of identical length to rice (Fig. 3). The use of alternative splice sites for exon 2 (166 bp from annotated AG and 76 bp downstream from annotated GT) would greatly improve similarity with predicted exon 2 from grasses. The predicted Arabidopsis exon 5 for gene 2 has six amino acids less than the predicted rice or barley protein at the start of exon 5. The use of an alternative splice site 18 bp downstream from the one that has been annotated in Arabidopsis would result in the addition of six amino acids (VPKVKQ) that are very similar to those found in rice and barley (VAKIKQ and VSKIKQ, respectively). These three modifications of the Arabidopsis annotation would improve the overall similarity between the Arabidopsis and barley predicted proteins from 72% to 75%. This alternative annotation is presented in Figure 3. The predicted gene structure in barley has been confirmed by the alignment of the predicted nucleotide sequence of the barley cDNA based on the genomic sequence with cDNAs from rye (BE494453, 96% identical), barley (BE413026, 99% identical), and maize (AI941761, 81% identical; TableI). The presence of this sequence in different cDNA libraries related to flowering may provide an indication of its function. The closest Arabidopsis homologue has been annotated as a putative cleavage stimulation factor subunit 1 (BAB10643).
Gene 3 is the largest of this interval with 790 amino acids in barley and 804 in rice. The translated proteins show 88% similarity (Table I) and are both divided in 18 exons, 14 of them of identical length (Fig.3). The closest Arabidopsis homologue (gene S1K1, 63% similarity with barley predicted protein, Table I) has 13 exons of identical length to rice and barley but has five additional predicted exons after exon 18. However, 30-bp downstream from the annotated donor splice site of Arabidopsis exon 18, a stop codon is present in the same position as in barley and rice. The 10 amino acids predicted for these additional 30 bp (CDTLRTILRL) are identical to the last 10 amino acids in rice and barley gene 3. The predicted amino acids from the additional five exons from the Arabidopsis protein produced no significant hits with the non-redundant and SwissProt databases, and therefore require further experimental confirmation. These last five exons are not included in Figure 3. The 18 exons predicted for barley gene 3 were confirmed by alignment of the predicted cDNA with different cDNA clones from sorghum (AW672216, 76% identical), maize (BE186368, 89% identical and BE475909, 88% identical), and rice (AU031105, 88% identical with barley and 98% identical with rice predicted cDNA) (Table I). The stop codon and the polyadenylation signal (409 bp downstream from the stop codon) were confirmed by alignment of the genomic sequence with maize EST BE475909 and rice EST AU031106 (97% identical with rice), respectively. The closest Arabidopsis homologue has been annotated as a Ser/Thr kinase with homology to yeast and mammalian stress activated kinases (AC008262).
The predicted barley protein from gene 4 showed a high similarity with the predicted rice protein (95% similarity) and with the closest Arabidopsis homologue (gene MXF12.6, 655 aa, 86% similarity, Table I). The rice, barley, and Arabidopsis genes are organized in 17 exons, 16 of which are of identical length in rice and barley, and 13 of which are identical in the three species (Fig. 3). The predicted gene structure was supported by the perfect alignment of the predicted rice and barley cDNA with six different overlapping cDNAs from rye, sorghum, rice, tomato (Lycopersicon esculentum), and pine (Table I). Gene 4 includes RFLP markers WG644 and R2311 (Harushima et al., 1998; Sarma et al., 1998; Kato et al., 1999). The closest Arabidopsis homologue has been annotated as an ABC transporter-like protein (BAB10828).
Barley genes 4a and 4b are duplications of an ancestral barley gene 4. The predicted 4a and 4b proteins have 17 exons of identical length (Fig. 3). The percentage of conserved amino acids between barley genes 4a and 4b (95%) is identical to that observed between barley gene 4a and rice gene 4 (95%) suggesting that this duplication is almost as old as the rice-barley divergence or that duplication has accelerated divergence between the two copies. Rice gene 4 showed lower similarity with barley gene 4b (92%) than with barley gene 4a (95%). This suggests that barley gene 4a may be either the true orthologue of rice gene 4 (if gene 4b from rice was deleted) or the barley copy that conserved a similar function to rice gene 4 (if the duplication never occurred in the rice lineage and the two barley copies diverged in their function).
The ends of exon 6 in barley genes 4a and 4b and rice gene 4 are not absolutely clear because the closest canonical GT 5′-splice sites force three different deletions/insertions that disagree with the boundary of homology between the three genes. The use of an alternative conserved GC 5′-splice site simultaneously eliminates the insertion/deletions in all three genes, determines exons of similar length with Arabidopsis and provides a perfect adjustment to the boundary of the barley-rice homology. This alternative GC 5′-splice site, predicted by comparative sequence analysis, was confirmed by the perfect alignment of the border between exons 6 and 7 with a rye cDNA (BE493856, BLAST score 829 E = 0). A similar non-canonical GC 5′-splice site has been reported in a comparison between the mouse and human GALT genes (Batzoglou et al., 2000).
BLASTN comparisons between rice and barley genomic sequences from gene regions successfully identified 123 of the 125 exons annotated in the colinear region. Almost no similarity was found in the intron regions, except a few base pairs flanking the exon-intron boundaries. The number of base pairs from each end of the significantly similar segments detected by BLASTN and the closest exon-intron boundary (including AG-GT splice sites) was calculated for the 208 exon-intron boundaries annotated in the colinear region. The average number of base pairs between these two points varied between 0 and 68 bp with an average of 9.8 bp and an sd of 11.8 bp. Once a larger number of comparisons between rice and barley genes become available, the distribution of these distances can be used to assign a probability to gene prediction programs based on rice-Triticeae comparisons.
Only exon 5 from gene 2 and exon 1 from gene 4 were not detected by BLASTN between the rice and barley genomic sequences. Exons 14 and 15 from gene 3 were detected but merged in a single segment by BLASTN. These four exons were detected by the gene prediction program GeneMark.hmm in both rice and barley. GeneMark.hmm (using the rice model) and Genescan (using the maize model) detected 56% and 22% of the 54 exons present in the rice genes. The number of exons predicted by these two programs in the barley genes (total 71 exons) was slightly higher than the number predicted in rice (GeneMark.hmm 62%, Genescan 32%), but significantly smaller than the number of exons detected by the rice-barley sequence comparison (98.4%).
Short stretches of homology between rice and barley were found upstream of the start codons of genes 1 and 3 using BLASTN. A region of 37 nucleotides (86% identical) was found 285 and 217 bp upstream of the start codon in rice and barley genes 1, respectively. Two conserved regions of 24 (94% identical) and 38 nucleotides (95% identical) were identified upstream of the start codons of gene 3 in rice (50 and 120 bp) and barley (41 and 91 bp). No significant homology was found between the promoter regions of barley genes 4a and 4b, or between any of the promoter regions in the grass genes and the closest Arabidopsis homologues. The conserved regions in genes 1 and 3 provide good starting points to delimit sequences with relevant biological function within the promoter regions.
Intergenic Regions
The organization of the different retroelements in these two BACs is shown in Figure 2. Coordinates for the different transposable elements are available in the annotated sequences in GenBank (AY013245and AY013246). The termini of retrotransposons and some terminal inverted repeat (TIR) elements such as Mutator (Bennetzen, 1996) are large enough and contain enough features to robustly ascertain exact insertion sites using only same sequence comparisons. However, other transposable elements are more variable in this aspect and coordinates for the boundaries required additional identification methods.
The larger distance between genes 1 and 2 in barley compared with rice is due in part to the insertion of two Copia-like retrotransposons and a non-long terminal repeat (LTR) retroelement (Ta, Tb, Tc; Fig. 2). These three transposable elements account for 18 of the 22-kb difference in size between the corresponding barley and rice intergenic region. The larger distance between genes 2 and 3 in barley compared with rice, similarly, is due to the insertion of a TIR element (Td, Fig. 2) and two BARE-1 retroelements (Te and Tf, Fig. 2). BARE-1-Tf is inserted into the 3′-LTR of theBARE-1-Te in opposite orientation (Fig. 2). A putative solo LTR of a BAGY-2 retrotransposon (Shirasu et al., 2000) was found upstream of the two nested BARE-1 elements. These elements account for 21 kb of the 29-kb difference between barley and rice in the region between genes 2 and 3. An additional 5-kb transposable element with homology to MuDR was found downstream of barley gene 4b (Tg, Fig. 2). EightStowaway miniature inverted transposable elements (MITEs) were found in the introns and intergenic regions of barley genes 3, 4a, and 4b.
One large Copia-like retrotransposon (6 kb) was found between rice genes 4 and 5 (Th, Fig. 2). A nonautonomousMutator transposable element and 11 MITEs were also found in this 50,000 bp of rice BAC 36I5.
Simple Sequence Repeats
BAC sequencing provides the opportunity to find SSR markers for specific regions of the genome. Simple sequence repeats (SSR), each with eight or more repeats, were identified in the rice and barley BACs (Fig. 2).
In rice BAC 36I5, one dinucleotide SSR was found in an intergenic region and two other dinucleotide SSRs were located within introns of genes 3 and 4. The long (TC)20 SSR located between exons 1 and 2 in gene 4 seems to be a good candidate to develop a PCR marker for this region of the rice genome by designing specific primers in the conserved flanking exons.
In barley BAC 635P2, two short dinucleotide SSRs were found in intergenic regions at the beginning and end of the BAC. The trinuclotide SSR located within the first exon of gene 2 is not a perfect repeat (GGC)3(GAC)(GGC)4 and no differences in size were found in this region between the barley cDNA and closely related cDNAs from rye (BE494453) and wheat (BE400296), suggesting that these repeats will not yield a polymorphic SSR.
Gene Discovery and Gene Structure
Sequence comparison between colinear rice and barley BACs 36I5 and 635P2 exemplifies the potential of comparative genomics as an independent method for gene identification to validate predictions made by gene finding programs (Avramova et al., 1996). Because no similarity was found between the intergenic regions, a visual comparison of the complete rice and barley BAC sequences using a dotplot immediately delimited the gene regions (Fig. 1). With the exception of rapidly evolving gene families (for instance, disease resistance genes; Leister et al., 1998), this method may provide a fast way to identify Triticeae genes once the complete rice sequence becomes available.
Sequence comparison was useful not only to identify the genes present in the region but also to provide valuable information for the determination of gene structure. Predicted gene structures were validated in most cases by comparisons with EST databases. A similar approach has been used in human-mouse comparisons of orthologous regions to accurately identify coding exons (Batzoglou et al., 2000). The percentage of exons identified by the BLASTN comparison of rice and barley gene regions (98.4%) was significantly higher than the percentage identified by GeneMark.hmm (59.2%) and Genescan (28%). However, four exons were correctly predicted by GeneMark.hmm and missed or merged by the rice-barley comparison, suggesting that a combination of gene finding programs, search of the EST databases, and comparative sequence analyses will provide the best strategy to determine gene content and structure.
None of the gene prediction programs were able to detect the non-canonical 5′-"GC" splice site at the end of exon 6 in gene 4. The alternative GC splice was first predicted by the comparative sequence analysis and then confirmed by the alignment of the predicted sequence with two different cDNAs. It would be useful to establish the frequency of these non-canonical sites in plant genes to assign them a correct probability in gene-finding programs.
If exon-intron boundaries were randomly distributed among the 3 bp of a codon, one-third of them would be expected to be between codons and two-thirds within codons. However, 66% of the observed exon-intron boundaries were located between codons and only 34% within codons (Fig. 3), indicating a non-random distribution. It is interesting to point out that the 16 exon-intron boundaries found within a codon were all conserved in Arabidopsis and both grass species, suggesting the possibility of some biological constraints in their modification.
Even though the comparisons between barley and rice genomic sequence were the most useful to determine exon-intron boundaries, comparisons with Arabidopsis homologues were useful to polish the gene annotation. Arabidopsis annotation can also benefit from the comparison with grasses. For instance, we found five regions (gene 1, exons 3 and 4; gene 2, exons 1, 2, and 5; and gene 3, exon 18) where data from the grass species suggested alternative annotations for the Arabidopsis genes that would increase similarity with the predicted grass proteins.
Gene Islands
The expected average distance between genes in barley is approximately 240 kb, based on the size of the barley genome (5,000 Mb;Arumuganathan and Earle, 1991) and assuming that barley has about as many genes (21,000) as predicted for Arabidopsis (Bevan et al., 1998). However, the average gene density in barley BAC 635P2 is approximately one gene every 20 kb, approximately 12 times higher than the expected genome average. A very similar gene density was observed in the sequence of two other chromosome regions in barley (Panstruga et al., 1998; Shirasu et al., 2000), suggesting that many barley genes are clustered in islands of higher-than-average gene densities. Gene clustering was also observed in several wheat regions (Feuillet and Keller, 1999; Lagudah et al., 2001), suggesting that this is a common feature of the large Triticeae genomes.
The existence of gene clusters was also suggested by studies based on preparative centrifugation in Cs2SO4 density gradients (Barakat et al., 1997). Barakat and coworkers (1997) designated these regions as "gene space" and suggested that they have a particular GC composition. However, the GC content of barley BAC 635P2 (44.2%) is outside the interval determined for barley "gene space" (45.8%–46.7%) according to Barakat et al. (1997). The GC content of BAC 635P2 is very similar to the average base pair composition of the barley (42.8%–45.6%) and wheat (45.7%) genomes (Melzer and Kleinhofs, 1987; Lagudah et al., 2001), suggesting that the base pair composition of the gene-rich regions is not significantly different from the average genome base pair composition in the Triticeae.
The base-composition of the 635P2 BAC (A = 27.6%, T = 28.2%, C = 21.8%, and G = 22.5%) is also very similar to the composition of the Copia-like retro-element BARE-1 (A = 25.8%, T = 28.1%, C = 21.4%, and G = 24.8%), which represents approximately 3% of the barley genome (Vicient et al., 1999). In wheat, 13% of the random sequences from 2 Mb of sheared T. monococcum genomic DNA showed significant similarity with sequences of a set of known Copia-like retro-elements (Lagudah et al., 2001). The large number of copies of these transposable elements suggests that their own base composition can have a large effect on the average base-composition of the complete genome.
Genome Size and Intergenic Regions
It is possible to speculate that the massive increase in genome size in the Triticeae may have also resulted in a tendency to have larger introns. However, the number of corresponding introns that were larger in barley than in rice was very similar to the number of introns that were larger in rice than in barley. Twenty-five introns were larger in barley and 23 were larger in rice. If the total length of the introns is considered, two genes were larger in rice and two in barley (Fig. 3). These two results suggest that most of the contributions to the differences in genome size between barley and rice occurred outside the genes. There was also a similar number of corresponding introns that were larger in rice than in Arabidopsis (27 introns) as vice versa (26 introns were larger in Arabidopsis), although the total length of the introns was larger in rice for the four genes analyzed here (Fig.3). These results further demonstrate that expansion and contraction of the introns was not affected by the differential expansion-contraction of the genomes outside of the genes but suggest that large insertions within the introns are more frequent or are retained longer in these grass species than in Arabidopsis. A larger number of genes should be compared to test this hypothesis.
The large difference in size between the rice and barley colinear gene regions was mainly due to the insertion of different layers of retroelements in the intergenic regions. Similar results were reported before in comparisons between colinear BACs from the small genomes of rice and sorghum with the colinear BAC from the larger genome of maize (Chen et al., 1997). The sequence of a 240-kb interval of the maize genome around the Adh1 gene (SanMiguel et al., 1996) showed 21 retrotransposons and two solo-LTRs, but only three small non-LTR retroelements (retroposons) and no large TIR elements (Tikhonov et al., 1999). The presence of a MuDR-related element in this 100-kb of barley DNA sequence may suggest a higher frequency of this element family in barley compared with maize (Bennetzen, 1996). However, larger regions should be compared to confirm this hypothesis.
As found in other studies (Bureau and Wessler, 1994; Tikhonov et al., 1999), most MITEs were associated with genes, inserting into introns or just upstream or downstream of them (Fig. 2). However, oneStowaway MITE was inserted into the 3′-LTR of retrotransposon Ta (Fig. 2). Such events appear to be relatively rare, and this is the first of which we are aware.
Cursory examination of the LTR sequences of the rice and barley retrotransposons discovered here suggests that deletions may be more common in rice than in barley. In the eight LTRs of the four intact retrotransposon of 635P2 only one insertion/deletion (indel) larger than 30 bp was found (corresponding to the insertion of the above mentioned Stowaway element into the Ta LTR). Yet, a large deletion of 521 bp and a 5-bp insertion/deletion were the only differences found between the sequences of the 36I5 retrotransposon's LTRs.
Rearrangement of the Rice, Barley, and Arabidopsis Genomes
Rice and barley have diverged for approximately 50 million years since they shared a common grass ancestor. During that time, a relatively small number of large chromosomal rearrangements have differentiated their nuclear genomes (Devos and Gale, 1997). However, these comparative mapping analyses have a 5- to 10-cM sensitivity that would miss most small chromosomal rearrangements. Our comparative sequence analysis of a four-gene segment indicates a single gene inversion and a gene duplication that differentiates these two genomes in this region. As this region represents less than 0.01% of either of these genomes, this suggests that thousands of such small re-arrangements may be present. From analysis of these two lineages alone, we cannot predict where or when these rearrangements occurred, but a broader characterization of other grass genomes should provide this information.
A comparison to the Arabidopsis genome suggests that genes from this region have been retained in the approximate 240 million years since these three species diverged from a common Angiosperm ancestor. Although none of the Arabidopsis gene pairs appear to be tightly linked, three of them are on the same chromosome, suggesting the possibility that most of the re-arrangements involved cis events like inversions, duplications, or deletions. A similar lack of colinearity between rice and Arabidopsis was previously reported by Devos et al. (1999). Despite differing only 3- to 5-fold in divergence time, Arabidopsis appears to have many fold more rearrangements relative to the grasses. This observation suggests that either most of these rearrangements occurred between the divergence times of Arabidopsis/grasses and barley/rice or that most of the rearrangements occurred specifically in the lineage that gave rise to Arabidopsis. Comparative genetic mapping studies have suggested that Arabidopsis may have had an unusually high frequency of chromosomal re-arrangement during evolution (Paterson et al., 1996, 2000), possibly a secondary outcome of its unusually small genome size.
BAC Selection
Colinearity between the long arm of rice chromosome 3 and the long arm of homeologous group 5 in the Triticeae has been studied extensively because of the presence of vernalization and frost tolerance genes Vrn-1 and Fr-1 in wheat and barley (Hordeum vulgare) and heading date geneHd6 in rice (Dubcovsky et al., 1998; Sarma et al., 1998;Kato et al., 1999; Sutka et al., 1999; Yamamoto et al., 2000). RFLP marker WG644 was mapped in this colinear region in rice, barley, and wheat (Kleinhofs et al., 1993; Dubcovsky et al., 1998; Sarma et al., 1998) and was used to screen the Morex barley BAC library (Yu et al., 2000) and the Nipponbare rice HindIII BAC library (http://www.genome.clemson.edu/orders/lib_desc/nippon.html). Positive BACs found in both libraries were fingerprinted with restriction enzyme HindIII to determine the approximate position of WG644 within the selected BACs. DNA was transferred to nylon membranes and hybridized with clone WG644 to confirm that all the BACs were from the same locus.
DNA from BACs 36I5 (rice) and 635P2 (barley) was extracted using the Large Construct Kit (Qiagen USA, Valencia, CA) and sheared with a HydroShear device (Genemachines, Inc., San Carlos, CA ) to two different average sizes of 2 and 9 kb. The sheared fragments were blunt-ended, dephosphorylated, and "A" tails added by incubation with Taq polymerase. Inserts were then ligated into pCR4TOPO using the TA cloning system. Clones were sequenced from both directions using big dye terminator chemistry and run on an ABI3700 capillary sequencer. Base calling and quality assessment were done using PHRED (Ewing and Green, 1998), assembled by PHRAP and edited with CONSED (Gordon et al., 1998). Gaps were filled by a combination of primer walking and shotgun sequencing of subclones with extremes at both sides of the sequencing gaps. Final error rate was estimated using CONSED.
Complete sequences from the rice and barley BACs were compared using the computer program Dotter (Sonnhammer and Durbin, 1995) to identify the conserved regions. Open reading frames were predicted using the rice training set of the gene-finding program GeneMark.hmm (Lukashin and Borodovsky, 1998;http://genemark.biology.gatech.edu/GeneMark/) and the maize (Zea mays) training set for Genescan (Burge and Karlin, 1997;http://genes.mit.edu/GENSCAN.html). Sequences from the BACs were also compared with NCBI dbEST and non-redundant databases using BLASTN, BLASTX, and TBLASTX algorithms (Altschul et al., 1997). Gene structure was determined by a combination of the two gene prediction programs mentioned above with GeneSeqer (Usuka et al., 2000;http://gremlin3.zool.iastate.edu/cgi-bin/prg/gs.cgi, maize splice site model) that generates splicing alignments of significant ESTs with the BAC genomic sequence.
Information from the gene-prediction and gene-splicing programs was combined with the identification of significantly similar segments between the barley and rice genomic sequence using BLASTN. These conserved regions, generally limited to the exons, were translated into proteins to adjust AG/GT exon-intron boundaries to the reading frames. The complete predicted cDNA and predicted protein were determined from the annotated structure of the gene. The predicted cDNA was aligned with the significant ESTs to confirm the annotated exon-intron boundaries. The predicted protein was used to search for the closest homologous (and, hence, possibly orthologous) Arabidopsis gene. The predicted Arabidopsis gene structure was then used to further polish the gene structure determined by the previous methods.
The positions of transposable elements were determined by a combination of FASTA, BLAST, and NETBLAST searches to the GenBank/EMBL nonredundant database and TIGR (http://www.tigr.org/tdb/rice/blastsearch.shtml), homology searches to known transposable elements, same sequence comparisons, and orthologous sequence comparisons. The sequences of both BACs were compared with themselves (same sequence comparison) and each other (orthologous sequence comparison) using COMPARE, DOTPLOT, STEMLOOP, REPEATS, and GAPs (Wisconsin Package Version 10.1, Genetics Computer Group, Madison, WI).
Restriction maps of 36I5 and 635P2 were constructed to experimentally validate computer sequence assembly. This experimental confirmation was important to determine the effect of large retroelements with large inverted repeats on the assembly algorithms. BACs were individually digested with 8-bp specificity restriction enzymes AscI, NotI, PacI,Pme I, and Swa I. All possible single and double digestions were analyzed for the restriction enzymes with one or more sites within this BAC. Restriction fragments were separated by pulse field electrophoresis in 1% (w/v) agarose gels (12°C, 14 h, 200 V, pulse 0.2–13 s). Gel filter replicas were hybridized with [α-32P]-labeled clones R2311, R2628, UCW12, Exons 1 to 3 from rice gene 2, exons 9 through 13 from rice gene 2, and BAC vector pBELOBAC11 (CVU51113). The two different segments from gene 2 were used to test the inversion of this gene.
The authors thank J. Emberton and D. Murphy for excellent technical assistance and G. Tranquilli and M. Helguera for preliminary work in the identification of the colinear BACs.
↵1 This work was supported in part by the National Science Foundation Plant Genome Program (grant no. 9975793) and by the U.S. Department of Agriculture National Research Initiative (grant no. 2000–1678 to J.D.).
↵* Corresponding author; e-mail maize{at}bilbo.bio.purdue.edu; fax 765–496–1496.
Received November 15, 2000.
Revision received December 14, 2000.
Accepted December 18, 2000.
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Plant Physiology Mar 2001, 125 (3) 1342-1353; DOI: 10.1104/pp.125.3.1342
Active Retrotransposons Are a Common Feature of Grass Genomes
Mutator Transposase Is Widespread in the Grasses
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