Genetics and Genomes (pp. 87-90 in Chapter 3. and all of Chapter 4). Mendelian Genetics (Eukaryotes) Review (will not be covered in class). I. MENDEL chose to study the inheritance of several characteristics of pea plants, each controlled by a single gene (single gene traits). His results led him to formulate a model with the following principles: A. GENES. Inheritance results from the transfer of particulate units. These are now called "genes" and we know that usually each gene encodes a particular polypeptide (protein) molecule. B. DIPLOID. Most sexually reproducing organisms live primarily in the diploid phase in which their cells contain two copies of each gene. Individuals within the same species differ because there are different forms of each gene. Different forms of gene X are called ALLELES of gene X. (As we will see, each allele results from a slightly different DNA sequence for the gene in question.) There may be many possible alleles for any gene, but a diploid cell or animal contains only two alleles of each gene or two copies of a single allele; a haploid gamete contains one copy of each gene, so only one allele. To study the inheritance of a gene, we must be able to identify at least two alleles which can lead to measurably different characteristics of the organism. The characteristic demonstrated by an organism due to its gene content is the PHENOTYPE. The gene content itself is the GENOTYPE. C. SEGREGATION (Mendel's 1st Law). Gametes (e.g., egg and sperm) contain only one of the two copies of each gene (haploid). Gametes result from a process (meiosis) in which the two copies of each gene are randomly separated ("segregated") so that the gametes receive one or the other but not both copies. Gametes have a 50-50 chance of receiving either copy of any given gene. Demonstrated by "monohybrid" (single gene) F2 crosses, see Fig.3.1. D. FERTILIZATION. A new organism results from the random joining of two gametes. Its cells again are diploid, containing two gene copies, one maternal and one paternal. Fig. 3.2. E. INDEPENDENT ASSORTMENT (Mendel's 2nd Law). Most genes (all the ones Mendel tested) segregate independently from one another. In other words, if a particular egg receives the "paternal" copy of gene A, it is still equally likely to receive the paternal or maternal copy of gene B. Demonstrated by "dihybrid" (two gene) F2 crosses, see Fig.3.3. [Mendel (unlike you) did not know about chromosomes and meiosis. The behavior of chromosomes during meiosis is the physical basis for Mendel's 1st and 2nd law (Fig. 14.30-14.34). In fact, Mendel's 2nd law is not strictly true for all genes, but is true for chromosomes. As we will see, genes which are closely linked on the same chromosome do not segregate independently, but chromosome pairs segregate independently during meiosis.] II. KEY TERMS TO UNDERSTAND: A. ZYGOSITY. A cell/organism which contains 2 identical alleles is HOMOZYGOUS for that gene. If the 2 alleles are different, the cell/organism is HETEROZYGOUS. B. DOMINANT/RECESSIVE. Consider a gene X for which there are 2 alleles: X and x. The genotype of a diploid organism could be XX, Xx, or xx. If the phenotype of XX is the same as Xx, we say that X (or its phenotype) is DOMINANT and x (or its phenotype) is RECESSIVE. If the phenotype of Xx is intermediate to XX and xx, the gene exhibits INCOMPLETE DOMINANCE. If the phenotype of Xx shows the properties of both those shown by XX and xx, the gene exhibits CODOMINANCE. C. A given gene may have MULTIPLE ALLELES. A given allele may be common or rare within a population of organisms, but any given allele can be present only in 0, 1, or 2 copies within any given individual member of that population.
WHY ARE SOME PHENOTYPES DOMINANT AND OTHERS RECESSIVE? WHY DO SOME PHENOTYPES "SKIP" A GENERATION? As you know, except for our germ cells (egg and sperm), we are DIPLOID organisms (each cell has two copies of every gene, other than genes on sex chromosomes, at least.) In most, but not all, cases one functional copy of a gene is sufficient for a normal phenotype. If the other copy is deleted or inactivated, the one good copy is sufficient, and there may be no change in phenotype. In this case, the deletion or inactivation mutation is RECESSIVE. It is a LOSS OF FUNCTION mutation, and almost all loss of function mutations are recessive. Most random mutations lead to a loss or decrease in function (because it's hard to improve on millions of years of evolution) and thus are recessive. Each of us probably carries several deleterious (perhaps even lethal) recessive mutations in our genomes without ever noticing it. Occasionally a genetic change occurs which causes the gene in question to do something new that it normally wouldn't (this could be good, bad or indifferent). This is a GAIN OF FUNCTION and most gain of function mutations are DOMINANT. A dominant trait will be expressed even if there is only one copy of the dominant trait gene (or dominant allele) in the cell. Note that a dominant lethal mutation (or sterile) mutation would rapidly be eliminated from any population, unless it produced its phenotype only after normal offspring-bearing years. Traits that are encoded by recessive alleles often "skip" one or more generations. Someone with a recessive trait must have two of the recessive alleles and therefore each of their offspring will get one. However, if their mate has two normal copies of this gene, all the offspring will get a normal copy and be normal (all will be heterozygous). If one of these heterozygous offspring mate with another person who is heterozygous for the trait, one fourth, on average, of their offspring (grandchildren of the first affected person) will show the trait. Generation "skipping" is even more common for sex-linked recessive traits, which are usually observed in males.
III. SEX DETERMINATION Sexual identity is usually determined by one or more genes on sex chromosomes. Males and females usually differ in the sex chromosomes they contain. 1. X and Y are used to describe sex chromosomes when (as in man and fruit flies) the female is homogametic (all gametes have identical sex chromosomes). In other words, the female is XX and all her gametes contain one X chromosome. The male is heterogametic (gametes have one or the other of two different sex chromosomes), XY, and half of his gametes contain one X and half contain a Y. 2. Chromosomes other than sex chromosomes are AUTOSOMES. 3. Generally, Y chromosomes have few genes. Their main function is to pair with the X during meiosis in male gamete formation. 4. Note that in XY organisms the male is HEMIZYGOUS (contains only one copy) for genes on the X chromosome. Thus, a recessive phenotype controlled by a gene on the X will be expressed much more often in males than in females. This is called SEX-LINKED INHERITANCE. IV. LINKED GENES, Fig. 3.3B A. Together, all the genes on a chromosome form a LINKAGE GROUP. B. Linked genes do not sort independently. However, the linked relationship between genes far apart on the same chromosome breaks down due to recombination events that occur between the two genes making it appear as if they are no longer linked (Fig. 3.4). Chromosomes break and rejoin with homologues at chiasmata, Fig 14.33, during meiosis leading to RECOMBINATION, Fig. 14.34. If not for recombination, all genes on the same chromosome would segregate together forever (i.e., not be assorted independently ). C. The further apart two genes are, the more likely a recombination event will occur between them. The closer two genes are together, the more tightly "LINKED" they are. So we can use the ratio of recombinant phenotypes to parental phenotypes to measure the genetic distance between two genes, for example, in a dihybrid cross. The distance between two genes as determined by the rate of recombination between them is the GENETIC DISTANCE or GENETIC LINKAGE DISTANCE. D. Sets of linked genes along with the linkage distance between them generate the GENETIC MAP of an organism, Fig. 3.5.
How do we know? 1. That genes are on chromosomes? Early in this century, Mendel's laws (having been essentially lost for about 30 years) were rediscovered and verified by several geneticists. One of these, W. Sutton, proposed that genes were on chromosomes, but had little evidence. At about this time, T.H. Morgan realized that the fruit fly, Drosophila melanogaster, was a particularly good experimental model. One of the earliest phenotypes he noticed was eye color: wild type eyes are red and he found a mutation leading to white eyes, which identifies the white gene. However, when he did a standard F2 mating starting with a white-eyed male and a wild type female, all the white-eyed F2 flies were male. In other words, the inheritence at the white gene was sex-linked (appeared to be linked to inheritance of sex-determining genes). At around the same time the cytogeneticists, N. Stevens and E. Wilson, were studying chromosome morphologies (karyotypes) in a variety of insects. For example, they noticed that grasshopper males have a single X chromosome (named for its unusual morphology) whereas females have two. The cytogeneticists realized that sexual phenotype could be determined by chromosome content, as described in III. above. Morgan then realized that linkage of the white gene to sex determination must prove it is on the X chromosome, confirming Sutton's hypothesis. 2. That each gene lies at a specific location on a chromosome and that genes on the same chromosome do not sort independently? Morgan quickly came across other sex-linked traits, mutants such as miniature wing (min) and yellow body (y). Both must lie on the X chromosome. When Moran did a dihybrid cross (such as Mendel's described in Fig. 3.3) between two of these genes, he got results that were clearly different from independent assortment (Fig. 3.3A) but also different from complete co-segregation (Fig. 3.3B). New recombinant genotypes were produced, which confirmed an earlier hypothesis of exchange of chromosomal material during meiosis by de Vries (i.e., recombination). Morgan showed that the level of recombination varied between different pairs of X-linked genes. Sturtevant went on to analyze Morgan's data, showing that they could be used to generate a linear, additive map of genes on the X chromosome.
V. GENOME: the complete array of all DNA within all the chromosomes of a given organism. Usually expressed as the haploid genome size for ease of comparison (Fig. 4.1). HAPLOID GENOME SIZES (TOTAL DNA PER HAPLOID CELL)
Common size range
Species genome size
100 to 100,000 Mb
1 Mb = 1 million base pairs. (Probably the number of essential genes does not differ greatly among various multicellular organisms. Most estimates are that humans have about 50-100,000 genes.)
EUKARYOTES PACKAGE LONG LINEAR DNA IN CHROMOSOMES
WHAT IS A CHROMOSOME? 1. Eukaryotic cells package their genetic information into a specific number of chromosomes. Diploid cells contain n pairs of chromosomes, for example, n for humans is 23, so diploid human cells contain 23 pairs of chromosomes (or 46=23x2). Haploid cells (generally gametes, i.e., sperm and egg cells) contain n chromosomes, or 23. A diploid human cell does not contain just any 46 chromosomes, rather it contains 2 copies of chromosome 1, 2 copies of chromosome 2, 2 copies of chromosome 3, etc. (the sex chromosomes, X and Y, are discussed later). A haploid human cell (sperm or egg) contains 1 copy of chromosome 1, 1 copy of chromosome 2, 1 copy of chromosome 3, etc. Each species has a specific haploid chromosome number, n. The specific set of chromosomes carried by a given cell is called its KARYOTYPE, Fig. 4.14. 2. Biochemically, chromosomes are primarily made of DNA (the genetic information) and PROTEINS which do the job of packaging the DNA, duplicating it, distributing it properly when cells divide, etc. 3. The pictures you see of chromosomes (two sausages tied together at one point) are MITOTIC CHROMOSOMES (Fig. 4.12), that is, chromosomes as they look in the tightly packaged form in which they exist during the time when cells are dividing. There are two sausages because at mitosis each chromosome has duplicated, so that each daughter cell will get one of them. Each sausage is a CHROMATID. The point at which the chromatids remain joined is called the CENTROMERE. The ends of each chromosome or chromatid are called TELOMERES. 4. Most of the time (INTERPHASE), chromosomes are less tightly packaged and so loosely distributed that we can't tell one from another. They fill the nucleus like a giant blob of spaghetti. (Fig. 4.11) 5. Each chromatid is a single, very long, linear DNA duplex, covered and condensed by chromatin protein. 6. Right after mitosis each chromosome is made of one chromatid. During the S (synthesis) phase of interphase, DNA is duplicated so that at the end of S, each chromosome is made of two chromatids joined together at the centromere.
VI. EUKARYOTIC GENOMES CONSIST OF VARIABLE AMOUNTS OF 3 CLASSES OF DNA, A. Single copy or unique DNA sequences: present once per haploid genome. (Contains most genes; prokaryotic genomes are almost entirely single copy DNA.) B. Moderately repetitive DNA: present in 10-1000 copies per haploid genome; e.g., genes encoding ribosomal RNA and some transposable elements (discussed later). C. Highly repetitive DNA: present in thousands of copies per haploid genome; e.g., short sequences tandemly repeated over and over that make up "constitutive heterochromatin": dense-packed chromatin at centromeres, telomeres, Y chrom., etc. Includes satellite DNA (Fig. 4.7).
How do we know? 1. The repetition frequency of different sequences in genomic DNA? (Fig. 4.6) This can be done by DNA hybridization kinetics, a technique pioneered by Roy Britten and Eric Davidson. Cellular DNA is isolated and broken into fragments (usually about 500 bp long). It is completely denatured (made single-stranded) by heating and then incubated under hybridization conditions. The more repetitive a sequence in the complex sample is, the higher the concentration of that sequence in the sample and the faster that sequence will find and hybridize to its correct complementary sequence. In this sort of experiment, prokaryotic DNAs, like E. coli, mostly hybridize in a single wave at a rate that suggests that the vast majority of prokaryotic DNA is single copy. Total eukaryotic DNA usually hybridizes in multiple waves that very roughly correspond to a rapid hybridizing fraction (highly repetitive), a slowly hybridizing fraction (single copy), and a fraction inbetween (moderately repetitive). Subsequent analysis using recombinant DNA techniques have shown that this is an overly simplistic viewpoint, but is still a useful generalization. It also varies significantly between species. For example, many species that have very large genomes compared to other of their class (corn vs. Arabidopsis in the genome size table above) have very high percentages of repetitive DNA in their genomes in comparison to their relatives with smaller genomes.
VII. GENOME MAPS: Genome maps allow us to understand the specific arrangment of genes and other sequences on each chromosome of a given species. This is important when we wish to relate one gene or landmark to another in the genome. Genome maps are of 3 major types. A. GENETIC: measures distances between genes by the RECOMBINATION FREQUENCY between Mendelian genetic markers, (Fig. 3.5) B. CYTOLOGICAL (or cytogenetic): measures distances between VISIBLE BANDS OR STAINED REGIONS found in chromosome KARYOTYPES, e.g., Drosophila polytene chromosome maps, Figs. 4.27-4.30, C. PHYSICAL: measures distances between genetic elements in terms of the LENGTH OF DNA between them. Fig. 4.23, 4.24 VIII. HUMAN GENETIC MAPPING A. POLYMORPHISM: In order to map genes, one must have at least two different alleles for any given gene and the two alleles must give rise to an observable phenotype. This difference is called POLYMORPHISM. Two of these polymorphic assays will be discussed further: B. RFLP, Restriction Fragment Length Polymorphism, Fig. 4.33 1. If one digests genomic DNA with any given restriction enzyme (RE) a complex mixture of thousands of fragments is produced. This mixture can be gel electrophoresed and an image of the gel pattern transferred to a Southern blot. The blot can then be hybridized with any cloned DNA probe and a pattern of radioactive bands will appear. 2. Sometimes the pattern differs (is polymorphic) between two individuals (or even between the two chromosomes in a single diploid individual). This is an RFLP. 3. RFLPs can be due to point mutations within the recognition site of the RE used or to point mutations within the fragment which give rise to a new site for that RE. They can also be due to insertions or deletions within the fragment being observed which would make the fragment larger or smaller. Often we cannot tell which of two alleles was the original type; we only know that there are two or more alleles. 4. Since RFLPs are part of the DNA sequence, they are transmitted as normal Mendelian markers. If we can identify both the maternal and paternal RFLP alleles, they will be inherited in a CO-DOMINANT fashion. 5. Some RFLP probes give many different bands and these complex patterns are often highly polymorphic. Such a probe can be used to "fingerprint" DNA. Finger-printing probes are often used in forensics. Other RFLP systems can detect mutations that cause genetic diseases; these RFLPs can be used for diagnostics. C. STSs (SEQUENCE TAGGED SITES, Fig. 4.38) A short region of DNA whose sequence is known, so that it can be amplified by PCR; may contain sequence polymorphisms. Many single base pair changes do not give rise to an RFLP. However, by amplifying the region in which that change exists using PCR methodology, one can individually assay such sequence changes in many individuals by automated DNA sequencing machines. DNA sequence analysis can be very powerful in diagnosing genetic disease, once we know the common DNA sequence changes that give rise to that disease (otherwise we don't know where to look among the millions of possible DNA regions for the change that causes disease). One particularly useful type of STS is the microsatellite marker (Fig. 4.34). A microsatellite is an STS which contains a tandem repeat of a very simple DNA sequence, e.g., (CA)n . Because errors are made in replicating such sequences the "n" often varies from one individual to another (i.e., it is polymorphic.) Since the value of n can be many possible numbers (multiple alleles) and varies a lot among the human population, it is an excellent genetic marker to make genetic maps and align them with physical maps.
How do we map genes in humans? 1. RFLPs. It was clear that human inheritance obeyed the rules of Mendelian genetics early in this century, but those interested in human genetics were very limited in what they could learn. Of course, one cannot design mating populations in humans and the number of offspring is a typical family is small. It was occasionally possible to show, for example, that a particular genetic trait or disease was inherited as a single gene locus, but one rarely, if ever, found multiple polymorphic traits in one family, so that linkage studies could be done. As described above, restriction enzymes had first been purified in the early 70's and the use of Southern blotting to analyze the restriction pattern of cloned genes became commonplace later that decade. David Botstein and Ron Davis at that time were working with yeast, and they knew that Southern blotting could be used to detect insertion/deletion mutations and mutations in restriction sites (it was not uncommon to notice such variation when labs cloned out different alleles of the same gene). In conversations with the human geneticist Mark Scolnick (and later Ray White), they realized this type of variation should provide powerful Mendelian markers that could be applied widely. Subsequent studies have shown that, any two humans (except identical twins) are really quite different in their DNAs (typically about 0.1 to 0.2% different; for comparison, humans differ from our fellow great apes by about 2% of our DNA base pairs). Most of those base pair differences have no observable consequence (i.e., no visible phenotype), but as more and more human genes were cloned (and more and more rest. enzymes became available), it became possible to map hundreds of RFLP in the single human pedigrees. The first complete human RFLP map appeared in 1987. 2. Microsatellites. While RFLP were a tremendous advance, doing all the Southern blotting required was tedious and costly. Furthermore, if one wishes to map a particular gene of interest in a particular family, it can sometimes be difficult to find any useful RFLP that is polymorphic in that family. Weber and May in 1989 (and a couple of other labs at about the same time) noticed a particular type of short tandem repeat (now called microsatellite or STR) often was found in and around human genes (see above). Since these are usually highly polymorphic and depend on PCR rather than Southern blotting (thus can be automated and tested much more cheaply), they have become the marker of choice in mapping human genomes.
IX. PHYSICAL MAPPING OF EUKARYOTIC CHROMOSOMES REQUIRES THAT WE BE ABLE TO HANDLE VERY LARGE DNAs (ca. 1 Mb). A typical human chromosome is on the order of 100 Mb so it would take over one hundred 1Mb fragments to cover it. The whole human genome is about 3000 Mb. It is extremely difficult to handle such large DNA fragments without breaking them and special techniques are also required to measure the size of such fragments and to clone them into recombinant DNA vectors. A. Sizing and Separating Large DNA Fragments: Large DNA fragments (up to 10 Mb) can be separated on special gels in which the direction of the electric field is altered in pulses (PULSED FIELD GEL ELECTROPHORESIS). (Fig. 4.22) B. Large Scale Restriction Maps: In order to produce specific large DNA fragments and to make large scale restriction maps, one needs restriction enzymes that cut DNA at sites which are very rare (the rarer the sites, the larger the fragments that are produced). This is usually done with restriction enzymes that recognize 8 base pair sites which, on average, will produce DNA fragments from 0.1 to 1 Mb. (Fig. 4.23) C. Cloning Large DNA: Large DNA fragments can be cloned in special vectors, such as YEAST ARTIFICIAL CHROMOSOME (YAC) VECTORS. The vector itself is a "minichromosome" made of a centromere and two telomeres with a cloning site and selectable markers. One can then insert very large DNA fragments (up to 1 Mb or more), transform the DNA into an appropriate yeast strain and the yeast will replicate the YAC as though it were an extra yeast chromosome. (Fig. 4.25, 4.26) X. How to correlate genetic, cytological and physical maps? A Cloned Gene or STS is the key. (Remember, any time you have an STS, you can always use PCR to make lots of that DNA.), See Fig. 4.35 for an integrated genetic, physical and cytological map of human chromosome 1. A. Physical maps are often restriction maps produced by overlapping cloned DNA fragments or Southern blotting using cloned DNAs as the hybridization probe. (See also Fig. 4.39) B. Genetic maps are produced using RFLPs deduced from Southern blots using cloned DNA as the probe or STSs using DNA sequence information derived by sequencing a cloned DNA fragment. C. Cytological maps are derived using in situ hybridization, again using a cloned DNA as the hybridization probe. XI. The utimate physical map is the complete sequence of the genome. (pp. 152-156) GENOME
l phage DNA
several other prokaryotes
E. coli chromosome
Chr III -1992, total 4/24/96
Human (23 chrom)
1st generation, Dec. 1993
1995 to present
Food for thought questions on the Human Genome 1. At one time, people with very severe genetic diseases did not live long enough or were not healthy enough to have children. With new techniques, including gene therapy, it is becoming increasingly possible for such people to reproduce. What do you think about the advisability of this? (One thing to remember. Most human genetic diseases are recessive and therefore the vast majority of transmission of these diseases is by heterozygous carriers who have no disease.) 2. Diagnostic DNA-based tests will be available in your lifetime for a variety of genetic-related diseases (propensity to cancer, diabetes, etc.). These tests could be performed long before the condition in question can develop. Should insurance agencies be able to require such tests and reject those with higher probabilities of disease? Should employers? Would you want to know the results of your tests (realizing that by modifying your nutrition and other habits you might be able to improve your chances; on the other hand, it could be a heavy psychological burden). 3. Genetic maps are measured in recombination units called centiMorgans (cM). On average in humans, there is about 1 cM for every Mb of DNA. However this ratio varies from one region of DNA to another and it even varies between meioses in males versus females. Why might this be?
Genetics and Genomes (pp. 87-90 in Chapter 3. and all of Chapter 4). Mendelian Genetics (Eukaryotes) Review (will not be covered in class). I. MENDEL ...
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4 Theoretical Biology, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands. 5 School of Life Sciences, Arizona State ... genomics leads to insights into the dynamics of selec- tively important variation and its ..... life-history stage
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