Monthly Archives: March 2017

TA cloning is a subcloning technique that avoids the use of restriction enzymes[1] and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together. PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3' end of the product. Such PCR amplified inserts are cloned into linearized vectors that have complementary 3' thymine overhangs.[2]

The insert is created by PCR using Taq DNA polymerase. This polymerase lacks 3' to 5' proofreading activity and, with a high probability, adds a single, 3'-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanines at the 5' end as this maximizes probability of Taq DNA polymerase adding the terminal adenosine overhang.[3] Thermostable polymerases containing extensive 3 to 5 exonuclease activity should not be used as they do not leave the 3 adenine-overhangs.[4]

The target vector is linearized and cut with a blunt-end restriction enzyme. This vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. It is important to use ddTTP to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end.[5] Manufacturers commonly sell TA Cloning "kits" with a wide range of prepared vectors that have already been linearized and tagged with an overhanging thymine .

Given that there is no need for restriction enzymes other than for generating the linearized vector, the procedure is much simpler and faster than traditional subcloning. There is also no need to add restriction sites when designing primers and thus shorter primers can be used saving time and money. In addition, in instances where there are no viable restriction sites that can be used for traditional cloning, TA cloning is often used as an alternative. The major downside of TA cloning is that directional cloning is not possible, so the gene has a 50% chance of getting cloned in the reverse direction.[1]

TOPO cloning

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TA cloning - Wikipedia

Calico cats are domestic cats with a spotted or particolored coat that is predominantly white, with patches of two other colors (often, the two other colors are orange and black). Outside North America, the pattern is more usually called tortoiseshell-and-white. In the province of Quebec, Canada, they are sometimes called chatte d'Espagne (French for '(female) cat of Spain'). Other names include brindle, tricolor cat, tobi mi-ke (Japanese for 'triple fur'), and lapjeskat (Dutch for 'patches cat'); calicoes with diluted coloration have been called calimanco or clouded tiger. Occasionally, the tri-color calico coloration is combined with a tabby patterning. This calico patched tabby is called a caliby.[1]

"Calico" refers only to a color pattern on the fur, not to a breed.[2] Among the breeds whose standards allow calico coloration are the Manx, American Shorthair, British Shorthair, Persian, Japanese Bobtail, Exotic Shorthair, Siberian, Turkish Van, Turkish Angora and Norwegian Forest Cat.

This condition arises when the individual cat has 2-X chromosomes. This is most common with females. However, rarely, a male cat is conceived with 2-X chromosomes (in addition to its Y chromosome). Because genetic determination of coat colors in calico cats is linked to the X chromosome, calicoes are nearly always female, with one color linked to the maternal X chromosome and a second color linked to the paternal X chromosome.[2][3] Because males only have one X chromosome, a male calico would have to have a rare condition where they have three sex chromosomes (two X chromosomes and one Y chromosome) in order to be calico. In addition to other symptoms caused by the condition, these male calicos are often sterile.

There is also a calico cat referred to as a Dilute Calico. Dilute Calicos are not necessarily rare. They are recognized by their grey, silver, and gold colors instead of the traditional white, black, brown or red patched coat of a calico. Dilute calicos are also called light calicos; because they usually have no dark colored fur.

The coat pattern of calico cats does not define any breed, but occurs incidentally in cats that express a range of color patterns; accordingly the effect has no definitive historical background. However, the existence of patches in calico cats was traced to a certain degree by Neil Todd in a study determining the migration of domesticated cats along trade routes in Europe and Northern Africa.[4] The proportion of cats having the orange mutant gene found in calicoes was traced to the port cities along the Mediterranean in Greece, France, Spain and Italy, originating from Egypt.[5]

In genetic terms, calico cats are tortoiseshells in every way, except that in addition they express a white spotting gene. There is however one anomaly: as a rule of thumb the larger the areas of white, the fewer and larger the patches of ginger and dark or tabby coat.[citation needed] In contrast a non-white-spotted tortoiseshell usually has small patches of color or even something like a salt-and-pepper sprinkling. This reflects the genetic effects on relative speeds of migration of melanocytes and X-inactivation in the embryo.[6]

Serious study of calico cats seems to have begun about 1948 when Murray Barr and his graduate student E.G. Bertram noticed dark, drumstick-shaped masses inside the nuclei of nerve cells of female cats, but not in male cats. These dark masses became known as Barr bodies.[7] In 1959, Japanese cell biologist Susumu Ohno determined the Barr bodies were X chromosomes.[7] In 1961, Mary Lyon proposed the concept of X-inactivation: one of the two X chromosomes inside a female mammal shuts off.[7] She observed this in the coat color patterns in mice.[8]

Calico cats are almost always female because the locus of the gene for the orange/non-orange coloring is on the X chromosome. In the absence of other influences, such as color inhibition that causes white fur, the alleles present in those orange loci determine whether the fur is orange or not. Female cats like all female placental mammals normally have two X chromosomes. In contrast, male placental mammals, including chromosomally stable male cats, have one X and one Y chromosome.[2][7][9] Since the Y chromosome does not have any locus for the orange gene, there is no chance that an XY male could have both orange and non-orange genes together, which is what it takes to create tortoiseshell or calico coloring.[citation needed]

One exception is that in rare cases faulty cell division may leave an extra X chromosome in one of the gametes that produced the male cat. That extra X then is reproduced in each of his cells, a condition referred to as XXY, or Klinefelter syndrome. Such a combination of chromosomes could produce tortoiseshell or calico markings in the male, in the same way as XX chromosomes produce them in the female.[citation needed]

All but about one in three thousand of the rare calico or tortoiseshell male cats are sterile because of the chromosome abnormality, and breeders reject any exceptions for stud purposes because they generally are of poor physical quality and fertility. In any event, because the genetic conditions for calico coloring are X linked, a fertile male calico's coloring would not have any determination in the coloring of any male offspring (who would receive the Y, not the X chromosome from their father).

As Sue Hubble stated in her book Shrinking the Cat: Genetic Engineering before We Knew about Genes,

The mutation that gives male cats a ginger-colored coat and females ginger, tortoiseshell, or calico coats produced a particularly telling map. The orange mutant gene is found only on the X, or female, chromosome. As with humans, female cats have paired sex chromosomes, XX, and male cats have XY sex chromosomes. The female cat, therefore, can have the orange mutant gene on one X chromosome and the gene for a black coat on the other. The piebald gene is on a different chromosome. If expressed, this gene codes for white, or no color, and is dominant over the alleles that code for a certain color (i.e. orange or black), making the white spots on calico cats. If that is the case, those several genes will be expressed in a blotchy coat of the tortoiseshell or calico kind. But the male, with his single X chromosome, has only one of that particular coat-color gene: he can be not-ginger or he can be ginger (although some modifier genes can add a bit of white here and there), but unless he has a chromosomal abnormality he cannot be a calico cat.[5]

It is currently impossible to reproduce the fur patterns of calico cats by cloning. Penelope Tsernoglou wrote "This is due to an effect called x-linked inactivation which involves the random inactivation of one of the X chromosomes. Since all female mammals have two X chromosomes, one might wonder if this phenomenon could have a more widespread impact on cloning in the future."[10]

Calico cats may have already provided findings relating to physiological differences between male and female mammals. This insight may be one day broadened to the fields of psychology, psychiatry, sociology, biology and medicine as more information becomes available regarding the complete effect of random X-inactivation in female mammals.[7][9][11]

Cats of this coloration are believed to bring good luck in the folklore of many cultures.[12] In the United States, these are sometimes referred to as money cats.[13] A cat of the calico coloration is also the state cat of Maryland in the United States.[14] In the late nineteenth century, Eugene Field published "The Duel", a beloved poem for children also known as "The Gingham Dog and the Calico Cat." In Japan, the Maneki-Neko figures depict Calico cats, bringing good luck.

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Calico cat - Wikipedia

John Heubusch is the classic "local boy makes good" story.

A House aide in the 1980s who later was the Labor Department's chief of staff during former President George H.W. Bush's administration, he went on to work for Gateway Computers, ran owner Ted Waitt's foundation and is now the executive director of the Ronald Reagan Presidential Foundation and Institute.

His next job: tackling the Second Coming.

Heubusch is the author of the just-released novel The Shroud Conspiracy, dubbed a DaVinci Code meets Indiana Jones thriller that considers the cloning of Jesus. It reads so well that a sequel has been ordered by Simon & Schuster imprint Howard Books, and Heubusch is in talks for a movie.

While he tapped his Washington roots researching the book, it is not a D.C. whodunit.

"When I tell people I've written a book and its sequel, to a person they all expect it's a Brad Meltzer-type novel given all my political experience, the Hill, the administration, lobbying. That's their expectation. But the subject matter I've chosen is far, far away from the halls of Congress," he said.

However, it does touch on a subject that is in the headlines. "One of the fundamentally important elements in the book is all about human cloning," Heubusch said. "From a research nugget standpoint, it's interesting to know that while human cloning seems to be outrageous, it's actually not outlawed in the U.S. There's no federal law prohibiting it."

At a book party for the Shroud Conspiracy a block from the White House Thursday evening, the author said that he has thought about the subject since he was 17, but added, "I don't really know what if feels like to be an author, but book was published just 48 hours ago."

Still, he added, it was already No. 30 on Amazon's best seller list.

Also from the Washington Examiner

"Shut your mouth," one audience member yelled.

03/19/17 3:35 PM

Paul Bedard, the Washington Examiner's "Washington Secrets" columnist, can be contacted at pbedard@washingtonexaminer.com

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Reagan library director taps bigger subject: Cloning Jesus - Washington Examiner

New York Times

Cloning Yo-Yo Ma: This Week's 8 Best Classical Moments New York Times In addition to reviews, features and news during the week, our critics and reporters collect the best of what they've heard: notes that sent shivers down their spines, memorable voices, quotations that cut to the heart of the story. Read the rest of ... and more »

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Cloning Yo-Yo Ma: This Week's 8 Best Classical Moments - New York Times