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Archive for the ‘Chromosome’ Category

DNA replication is carried out by DNA polymerase. During the replication, one DNA strand synthesized as leading strand and the other as lagging strand. The leading strand synthesis is continuous, whereas lagging strand synthesis is discontinuous. DNA replication at the DNA ends i.e. telomeres is an altogether different story. If a primer forms at the end of a linear DNA molecule to initiate replication of the leading strand, a single-stranded gap remains at that end after DNA has been synthesized and the primer has been removed. DNA pol cannot fill his gap as no 3-OH group is available onto which nucleotides can be added. If this gap were left unfilled a linear DNA molecule would shorten at each end by the length of the primer with each replication cycle. After many rounds of replication, the molecule would disappear. This problem is known as the linear DNA replication paradox. Besides, as the lagging strand is replicated in the discontinuous manner, it cannot be replicated in its entirety. When the RNA primer is removed, there is no upstream strand onto which DNA pol can build to fill the resulting gap. Without some special mechanism, the daughter DNA strand resulting from lagging strand synthesis would be shortened from the telomeric ends at each cell division.

The enzyme that prevents this shortening of DNA strands is known as telomerase. The enzyme functions as a reverse transcriptase (RNA-dependent DNA polymerase). It holds a small RNA molecule as its component. It was discovered by Elizabeth H. Blackburn et al. The enzyme contains a catalytic site that polymerizes the nucleotides using RNA as the template. The RNA template is part of the enzyme and is approximately 160 bases long. The enzyme uses this RNA as template for adding telomeric repeats to the chromosome ends. First, the 3’ end of the telomere hybridizes with the RNA component of telomerase. Within the RNA sequence is the 6-nucleotide repeated sequence 5’CCCCAA3’ (Tetrahymena) which is complementary to the telomere repeat sequence. Telomerase binds to the 3’ end of to the repeated region. Telomerase binds to the 3’ end of the ssDNA, adding new copies of the TTGGGG repeat sequence. These repeats further extend the single stranded gap left after primer removal. Primers may be added to the repeated segments of DNA and a DNA pol may now fill in much of the gap with DNA.

The repetitive sequence added by telomerase is determined by the RNA associated with the enzyme, which differs among telomerases from different organisms. This extends the dsDNA till it is at least as long as, or longer, than the parent molecule. The single stranded end left after several rounds of addition can be trimmed. The extension and trimming of telomeric DNA is not precise and therefore the number of telomeric repeats is highly variable. Certain proteins, like Rap1 in S. cerevisiae, TRF1 in human beings, keep track of the number of telomeric repeats. It is actually the number of protein molecules attached to the telomere ends that is counted, and not the number of repeats, to decide if telomeres should be added.

The activity of telomerase gradually declines indicating that the DNA molecules must grow shorter with each cell division. This is of little consequence for most cells as they undergo a limited number of cell divisions and then stop dividing: This is the normal ageing process which occurs in all body cells, end at a certain telomeric length, and cells stop dividing. However, if telomerase starts behaving erratically, the chromosomes will not be shortened and cells will continue to divide leading to continuous growth. When cancer cells were examined for the presence of telomerase, the active enzyme was found in abundance, indicating that the linear DNA molecules were being fully replicated. Thus telomerase is found to be essential for cancer cells to continue dividing. Thus, the attention is now turning to the possible clinical application of this knowledge. Research is now under way to determine if blocking the activity of telomerase may be an effective treatment for cancer. Further, studying normal telomere shortening, which acts as a biological clock, may help understand senescence.

 


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This post is about two recent developments in the field of biotechnology and environmental science.

1) Agrobacterium-mediated transformation is the method of choice for inserting foreign genes into the plant genome. Through this method one gene could be introduced at a time in a single experiment. However, to introduce two foreign genes, one needs to develop two separate plant lines each carrying one gene. The plants of the two lines are then cross bred to get both the genes in one plant. The process is quite laborious and takes a lot of time to complete. Besides, integration of the foreign gene into the plant genome can disrupt any native gene; or the foreign gene may get silenced and not expressed. To overcome these problems a team of scientists from the University of Chicago, the University of North Carolina and Chromatin, Inc., has developed a new method for constructing artificial plant mini-chromosomes. These are the rings of plant DNA that can be used to carry multiple genes in plant cells in one go. The work has been published in the October 19, 2007, issue of PLoS-Genetics. The team has developed maize mini-chromosomes (MMCs) that “…can introduce an entire “cassette” of novel genes into a plant in a way that is structurally stable and functional.” These mini-chromosomes could be suitably used as vectors for the transfer of two or more than two genes simultaneously, and would thereby save the time and effort required for lengthy and laborious hybridization experiments needed to transfer two genes in a plant genome. This would also cut down expenses involved in such experiments. The MMCs created also have maize centromere sequences in them. These sequences help them maintain independently of the maize genome and  newly introduced genes remain separate from the genome. This also helps their efficient transfer to the next generation in the Mendelian fashion. Thus these MMCs behave much like ordinary chromosomes.

You can read the full story here.

2) The second story deals with the use of algae as biofuel. Several companies are already working on the use of algae as biofuels. But LiveFuels Alliance, funded by LiveFuels Inc based in Menlo Park, CA, is trying to tap the oil producing potential of algae and hopes to replace gallons of fossil fuels with algae-based biocrude by 2010. Algae synthesize oil naturally. The raw algae is processed to “…make biocrude, the renewable equivalent of petroleum, and refined to make gasoline, diesel, jet fuel, and chemical feedstocks for plastics and drugs.” To quote from the story, “Theoretically, algae can yield between 1,000 to 20,000 gallons of oil per acre, depending on the specific strain.” What is important is that LiveFuels Alliance is a national initiative led by Sandia National Laboratories, a U.S. Department of Energy National Laboratory, and in the next 3 years it would sponsor several labs and hundreds of scientists, which makes it the largest endeavor focused on commercial biofuel production from algae. Read the whole story here. via: Inhabitat

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Centromere contd…

The sequence elements of Saccharomyces cerevisiae centromeres are not present in other eukaryotes; they are absent even in other yeast species. The DNA sequences of centromeres are highly diverse among eukaryotes. However, some similarities have been revealed in centromere consensus sequences among eukaryotes. The centromeres of multicellular eukaryotes are surrounded by highly repetitive DNA ; centromeres themselves contain tandem repeats. One of the centromere repeat sequences present in humans is called alphoid, also known as α-satellite DNA. It is about 170 nucleotides in length and is repeated many times in a tandem array in the centromere.

Alphoid sequences have a conserved sequence called the CENP-B box. It has the following consensus sequence:

PyTTCGTTGGAAPuCGGGA, where Py=pyrimidine and Pu=purine.

This sequence serves as a binding site for proteins associated with the centromere. The CENP-B box is also found in other mammals and in Drosophila. The CENP-B box may be a common sequence in the highly diverse centromeric sequences of multicellular eukaryotes.

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Centromere

The constricted region on the chromosome where the two DNA molecules are attached is called the centromere. Much of the sequence information available on centromere is based on comparison of centromeric DNA sequences among the chromosomes of Saccharomyces cerevisiae or the budding yeast. Each of the yeast chromosome carries contains a conserved centromeric region called CEN, which is similar among all the 16 chromosomes. The CEN region consists of about 160-220 nucleotide pairs that have been divided into three elements called CDEI, CDEII and CDEIII.

CDEI is an 8-nucleotide-pair sequence with the following consensus sequence:

A/CTCACA/CTG

CDEI, although important for proper centromere function, is not essential. The deletion of this sequence does not eliminate mitosis; however, chromosome stability declines during mitosis.

CDEII is a sequence of 78-86 nucleotide pairs which are A-T rich: They contain about 90% A-T pairs. CDEII is important for centromere as its deletion causes a loss of some chromosomes during cell division.

CDEIII is a 26-nucleotide-pair element with the consensus sequence

TGTTTTTGNTTTCCGAAANNNAAAAA

where the sequence highlighted in red (bold) is palindromic. CDEIII is essential for centromere function. If a substitution mutation alters the cytosine (highlighted in green) in the center of the palindrome, the centromere no longer functions.

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This post is in continuation to the previous post Telomeres: The ends of the chromosome

Once telomeres have been added to the chromosome ends, different organisms use any of the three different methods to protect the ends of chromosomes.

1) The ends of telomeres which are guanine-rich form complex structures called the G-tetraplex. Four Gs form this planar structure in which they are hydrogen bonded to each other.

2) Some proteins are found to bind to the 3′ ends of telomeres. In the ciliate oxytricha nova, a protein called the telomere-end binding protein (TEBP) attaches to the 3′ ends of telomeres and protects them.

3) A third structure known as t-loop has been discovered at the end of mammalian telomeres. This loop forms at chromosomes ends under the influence of a protein called TRF2 (telomere repeat-binding factor), which causes the 3′ end of the chromosome to loop around and interdigitate into the double helix, forming the loop.

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Telomeres define the chromsosome ends. The two criteria which identify a telomere are:

1) It must lie at the chromosome end.

2)  It must confer stability on a linear molecule.

Several telomeric sequences are known from various eukaryotic organisms. Each telomere consists of a long series of short, tandemly repeated sequences. The telomere construction follows a universal principle. All of the telomeric repeat units are written in the general form Cn(A/T)m, where n>1 and m is 1-4.

Plants have C3TA3 and humans have C3TA2 basic telomere repeat sequences/units. The number of copies of the basic repeat unit in telomeres varies across species, from chromosome to chromosome within a species, and even on the same chromosome at different stages of the life cycle.

In some species, the telomeres terminate with a single stranded region of the DNA strand with the 3′ end (so-called overhang). Terminal bases of this single-stranded end exhibit unique patterns of methylation that probably contribute to the formation of a unique hairpin or folded structure at the tip of the telomeric DNA. Additional repetitive DNA sequences-referred to as telomere-associated sequences-are present adjacent to the telomere.

   

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The Chromosome

The term chromosome was coined by W. Waldeyer in 1888. The word chromosome means “coloured body”. Chromosomes were discovered by C. von Nageli in 1842. Each chromosome is composed of proteins and nucleic acid (DNA). The proteins in case of higher organisms are histones. Thus chromosomes are nucleoprotein in nature. The nucleoprotein material of the chromosome is referred to as chromatin. The chromatin component is present in two states: euchromatin and heterochromatin. Chromosomes are usually diagrammed in their most condensed state (during the metaphase of cell division). The metaphase chromosome contains two DNA molecules because the DNA of each chromosome replicates before the cell divides.  The two DNA molecules  are attached at a constricted site on the chromosome called the centromere. There are present protein bodies called kinetochores on the either side of the centromere. The kinetochores participate in separation of the replicated chromosome during mitosis.

The constricted centromere is often located near the middle of the chromosome. But the centromere may also be present at locations different form the middle of the chromosome. On the basis of centromere location, chromosomes can be metacentric (centromere in the middle), submetacentric (centromere located somewhat off centre), telocentric (centromere at the chromosome end), subtelocentric or acrocentric (centromere near the end). The term acrocentric is used when describing human and animal chromosomes; subtelocentric is used for describing plant chromosomes. There are no telocentric human chromosomes.

The centromere is also called the primary constriction. Occasionally, another constriction, called a secondary constriction is present near one end of the chromosome. Beyond the secondary constriction a small portion of the chromosome extends into a chromosome satellite. At these regions is found the nucleolus organizer region (NOR), where rRNA genes are clustered.

When the DNA in the centromere is duplicated, the two short and two long arms of the chromosome are attached to a single centromere. The term chromatid denotes the identical duplicated portions of the chromosome. The two chromatids of a chromosome are called sister chromatids, and each sister chromatid has one linear DNA molecule. The centromere divides the chromosome into segments called chromosome arms. It divides the chromosome into a long arm and a short arm except in the case where centromere lies in the middle. In metacentric chromosomes where centromere lies at the centre, the distinction between long and short arms diminishes. The short arm is called p arm (p=petit); the long arm is q arm.

The ends of eukaryotic chromosomes are specialized and are known as telomeres. Telomeres stabilize the ends of the chromosome, and prevents other chromosomes/chromosome fragments from fusing to it. Telomeres also protect the end of the DNA in the chromosome from enzyme degradation.

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