Chromosome and Genomic Organization 1 PDF

Chromosome and Genomic Organization 1  Types of DNA Sequences in Eukaryotes Indications that the DNA of eukaryotes contains several types of sequences came from the results of studies in which double stranded DNA was separated and then allowed to reassociate. If double stranded...

Chromosome and Genomic Organization 1  Types of DNA Sequences in Eukaryotes Indications that the DNA of eukaryotes contains several types of sequences came from the results of studies in which double stranded DNA was separated and then allowed to reassociate. If double stranded DNA in solution is heated, the hydrogen bonds that hold the two strands together are weakened and at a certain temperature, the two nucleotide strands separate completely (denaturation of DNA). The denaturation of DNA by heating is reversible. If this single stranded DNA is slowly cooled, hydrogen bonds will again form between complementary base pairs, producing double-stranded DNA (renaturation of DNA). During renaturation, single DNA strands associate randomly with their complementary strands and hydrogen bonds form between complementary bases. In a typical renaturation reaction, DNA molecules are first sheared into fragments several hundred base pairs in length. Next, the fragments are heated to about 100°C, which causes the DNA to denature. The solution is then cooled slowly, and the amount of renaturation is measured by observing optical absorbance. The amount of renaturation depends on two critical factors: 1) initial concentration of single-stranded DNA (C0) 2) amount of time allowed for renaturation (t). A plot of the fraction of single-stranded DNA as a function of C0t during a renaturation reaction is called a C0t curve.  Unique Sequence DNA Unique sequence DNA refers to sequences that are present only once, or at most a few times, in the genome. Unique sequence DNA includes sequences that code for proteins as most proteins in eukaryotic cells are encoded by genes present in one or a few copies. Repetitive DNA Repetitive DNA consists of moderately repetitive and highly repetitive DNA sequences that appear many times within the genome. The sequences can be distributed in the genome as 1) distributed at irregular intervals (dispersed repetitive DNA) 2) clustered together so the sequence repeats many times in a row (tandemly repeated DNA)  Centromeric DNA Sequences Centromeres play a critical role in separation of chromosomal homologues during mitosis and meiosis. The DNA sequence contained within the centromere is vital for this role. Within this region of the chromosome, the DNA associates with the kinetochore, a complex of proteins associated with the centromere during cell division, to which the microtubules of the spindle attach. Centromeric sequences of eukaryotes vary considerably in size. The DNA found in the centromeric regions of chromosomes is composed of short repetitive sequences, differs from main band DNA in its molecular composition and forms a separate satellite band in an ultracentrifuge density gradient (satellite DNA). Separation of main band (MB) and satellite (S) DNA from mouse by ultracentrifugation in a cesium chloride density gradient.  Telomeric DNA Sequences Telomeric DNA sequences consist of short tandem repeats and are required for replication and stability of a linear chromosome. Simple telomeric sequences are present at the extreme end of chromosomal DNA molecules. Depending on the organism and its stage of life, the number of copies of repeats varies between 100-1000. In all vertebrates, including humans, the sequence 5’-TTAGGGG-3’ is repeated multiple times. Telomere associated sequences are regions internal to the simple telomeric sequences and contain repeated complex DNA sequences. An enzyme, telomerase is involved in the replication of these repeat units. Telomerase is a member of a class of enzymes called reverse transcriptases, which are used in specialized situations to synthesize DNA from RNA. The additional DNA is then able to act as template for synthesis on the lagging strand. This process counteracts the tendency to shorten at replication. An age-dependent decline in telomere length has been found in cells and tissues.  Middle Repetitive Sequences In addition to the highly repetitive DNA sequences, a second category of repetitive sequences, moderately (middle) repetitive DNA, is also present. Variable Number of Tandem Repeats (VNTRs) A class of tandem repeats shows variable number at different chromosomal positions and in different individual members of a species. This type of repeat is called a variable number tandem repeat or VNTR (sometimes called minisatellite). The VNTR loci in humans are sequences consisting of variable numbers of a repeating unit from usually from 15 to 100 bp long. Many such sequences are found throughout the genome. Because of the variability in the number of tandem repeats from individual to individual, VNTRs can be utilized for DNA fingerprinting. Short Tandem Repeats (STRs) Another group of tandem repeats consists of repeating sequences 2-6 nucleotides in length. This type of repeat is called short tandem repeat or STR (sometimes also called microsatellite). Like VNTRs, STRs are dispersed throughout the genome, vary among individuals in the number of repeats present at any site and can be utilized for DNA fingerprinting and as molecular markers for genome analysis  Repetitive Transposed Sequences Another category of repetitive DNA consists of sequences that are distributed individually throughout the genome instead of being tandemly repeated. These can be short or long and have the added distinction of being transposable elements which are mobile and can potentially move to different locations within the genome. Short Interspersed Elements (SINEs) are usually between 100 - 300 base pairs in length. The best characterized human SINE is a set of closely related sequences called the Alu family. Members of this family, also found in other mammals, are 200-300 base pairs long and are dispersed rather uniformly throughout the genome both between and within genes. In humans, this family comprises around 5% of the entire genome. SINES are transposons and have the potential for rearrangement within the genome. However, SINES do not encode the enzymes they need for movement but can move if these enzymes are supplied by an active LINE transposon. Long Interspersed Elements (LINEs) represent another category of repetitive transposable DNA sequences. LINES are usually between 4-6 kb in length. The most prominent example of LINES in humans is the LINE-1 (L1) family. Members of this sequence family are about 6 – 7 kb long. Their 5’ end is highly variable and the role within the genome is yet to be defined. LINES are transposons and can move from location to location within the genome. Genes they encode contain enzymes necessary for such movement. A typical mechanism of transposition of L1 elements is: The L1 DNA sequence is first transcribed into an RNA molecule. The RNA then serves as the template for synthesis of complementary DNA by means of the enzyme reverse transcriptase (encoded by a portion of the L1 sequence). The new L1 copy then integrates into the DNA of the chromosome at a new site. Due to the similarity of this transposition mechanism with that utilized by retroviruses, LINEs are sometimes referred to as retrotransposons. Middle Repetitive Multiple Copy Genes In some cases, middle repetitive DNA includes functional genes present in multiple copies. For example, many copies exist of the genes encoding ribosomal RNA with around 120 copies present in the Drosophila haploid genome. Single genetic units encode a large precursor molecule that is processed into the 5.8S, 18S and 28S rRNA components. In humans, multiple copies of this gene are clustered on the p arm of the acrocentric chromosomes 13,14,15, 21 and 22. Multiple copies of the genes encoding 5S rRNA are transcribed separately from multiple clusters found on the p arm of chromosome 1.