MCB 201 Gene Expression - Spring Semester 2003
Lecture 9 (Molecular Structure of Genes and Chromosomes)
Section 9.2 continued (Chromosomal organization of genes and noncoding DNA)
1. Figure 9-6. Use of simple-sequence DNA as chromosomal marker. In this figure, all of the human chromosomes in this 'metaphase spread' were uniformly stained with a fluorescent dye. Then they were hybridized with a specific simple-sequence DNA labeled with a fluorescent reporter molecule called biotin, which appears as a yellow band on chromosome 16, thus locating this particular simple sequence to one site in the genome.
2. Figure 9-8. Consensus sequences of the repeat unit of human minisatellites named lambda33.1 (62 bp repeat) and lambda33.5 (17 bp), based on analysis of more than ten sets of repeats in each case. Even small differences in the total lengths (due to different numbers of repeats of the sequence) of various minisatellites from different individuals can be detected. Red letters indicate positions where base differences were detected among the ten sequences. Red dot indicates a base deletion. Minisatellites are repeated in relatively short regions of double-stranded DNA, 1- 5 kb long, containing about 20-50 repeats of the basic sequence which can be 15-100 bp long. Note that the lengths of some simple sequence repeats are unique to each individual in a mammalian species, providing the basis for DNA fingerprinting. Within a species, the nucleotide sequences of simple sequence DNAs are highly conserved (very similar).
3. Figure 9-9. Human DNA fingerprints. In this gel, DNA samples from three individuals (1,2 and 3) were subjected to Southern blot analysis using the restriction enzyme Hinf1 and three different radioactively labeled minisatellites (a,b, and c) as probes.
Section 9.4 (Functional Rearrangements in Chromosomal DNA)
Functional rearrangements in DNA regions occur in both eukaryotic and procaryotic cells by mechanisms involving DNA inversion, deletion and amplification of DNA. Functional DNA rearrangements represent a mechanism for regulation of gene expression of a small number of genes, but the consequences of this kind of regulation can be very important.
4. Figure 9-20. Control of expression of Salmonella flagellar proteins (H1 and H2) by DNA inversion. Salmonella typhimurium is a pathogenic bacterium causing food poisoning. Two different types of proteins that assemble into bacterial flagella can be produced by Salmonella, but only one gene is expressed at a time. The switch from expression of one of these proteins to expression of the other is called phase variation. It is infrequent, but occurs much more often than random mutational events, and so it can be distinguished from these. The two ways that this segment of DNA can be expressed are shown in this diagram. In Phase I, the promoter is oriented in the appropriate direction to allow transcription of the H2 and rH1 genes. The H2 gene produces the H2 flagellar protein and a repressor protein rH1. This repressor binds to the regulatory region of the H1 gene and inhibits transcription of this gene. In Phase II configuration, the promoter is inverted and is now in the opposite direction, essentially inactive, and so the H2 gene and rH1 gene are not transcribed. As soon as the rH1 repressor levels decline in the cells by degradation or dilution during cell division, the H1 gene can then be transcribed and H1 flagellar protein is made. The consequence for the immune system of an infected person, is that antibodies are first raised to the H2 flagellar antigen, and then a few cells switch to H1 flagella and the antibodies to H2 cannot neutralize these bacteria. The body must then mount a second immune response which requires several more days to produce anti-H1 antibodies to neutralize the infection.
5. Figure 9-21. Inversion of H2 promoter mediated by a site-specific recombinase called Hin, a dimeric protein. The inversion of the H2 promoter gene region is carried out by an enzyme called a site-specific recombinase which is encoded by the Hin gene, conveniently located within the inverting segment of DNA. The Hin protein binds at each of two sites flanking the H2 promoter, dimerization of Hin protein brings together a loop of DNA, the strands of which are sequentially cut and ligated to the opposite strand. This is called site-specific recombination or crossing-over, because the DNA strands are crossed in the process. The gene encoding Hin is not expressed very often, thus the low frequency of phase variation.
6. Figure 9-22. Schematic model of an IgG antibody molecule showing its domain structure. We have a type of cell in our bodies called B lymphocytes that are capable, as a population, of producing millions of different kinds of antibody molecules. All of these antibodies are encoded in genes. How is this large repertoire of genes produced? Before we answer this question, let us first look at the product of these genes, antibody molecules. Each antibody molecule of the IgG class is composed of two 'light chains' and two 'heavy chains', so named because the polypeptide chains are of different lengths.The antigen binding site is the most variable part of these types of molecules in amino acid sequence, and both heavy and light chain sequences contribute to the binding site. Each B cell can be stimulated by an antigen to differentiate into a plasma cell that produces and secretes one kind of antibody molecule.
7. Figure 9-23. Organization of the k light-chain locus in human germ-line DNA. Since the human genome only has about 30,000 genes (according to the Human Genome Project 2001), how are the millions of genes needed to produce our antibody repertoire generated? These genes are not in the human germ line (i.e. sperm and egg DNA). In fact, they are created during B cell differentiation from stem cells by mechanisms involving inversions and deletions of DNA regions (even so-called 'identical' twins are not the identical genetically). In order to understand how these genes are created during development, we need to first understand the organization of the DNA regions used to produce these genes, as it exists in the human germ line DNA. The organization of these regions is shown here for a light chain family called the kappa light chains (I will leave the 'k' designation off of the V, L, and J region abbreviations in this discussion). Note the many copies of the L and V segments. L DNA encodes the signal sequence on the light chain that is needed to target this polypeptide chain into the rough endoplasmic reticulum (ER) of cells. Remember that antibodies are secreted proteins and are synthesized on ribosomes associated with the ER and then processed through the secretory pathway of plasma cells for release into the blood, etc. The V segment will contribute sequence information to produce the variable domain of the antibody molecule. At the end of the V segment is a sequence (shown as a red box) involved in joining with a J segment. The J segments have two functions. They are involved in joining the V segment to the Ck segment and they also contribute sequence information to produce the variable domain. The Ck segment encodes the sequence information needed to produce the constant domain of this light chain.
8. Figure 9-24. Joining of V to J in human germ-line DNA and formation of a k light chain. Part of what creates the diversity of light chain genes is the random joining of V regions and J regions. Although this diagram looks complicated, the joining is relatively straightforward. It requires a site-specific recombinase that recognizes sequences at the 3' end of each V segment and the 5' end of each J segment. Recombination between these segments results in either a deletion or inversion of the intervening sequence, depending on the transcriptional orientation of the L+V unit relative to the J segment. Direction of transcription is shown by the small arrows drawn parallel to the DNA. Note that deletions occur with the transcriptional orientation of the segments being joined are in opposite directions, and inversions occur when they are both oriented in the same direction. An additional mechanism for generating diversity is the random loss of nucleotides from the joining site. As you might expect, this causes a shift in the reading frame two-thirds of the time and essentially scrambling of the code for producing a functional polypeptide chain. But, apparently the generation of antibody diversity is more important and the waste in this process has been tolerated during evolution.
9. Figure 9-26. Model of DNA amplification to produce polytene chromosomes. The salivary glands of Drosophila contain very large cells and perhaps to make sufficient protein, they have large chromosomes known as polytene chromosomes. These chromosomes are produced by many rounds of DNA replication in which the progeny strands do not separate. Instead they are held in parallel alignment, as many as 1024 strands, all potentially active in gene expression. Not all of the chromatin is amplified in this way. Simple sequence regions remain single-copy and ribosomal RNA genes are not amplified as much as other parts of the chromosome.
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