BIO 345 Introductory Genetics

BIO 345 Introductory Genetics
Review Items for First Exam Spring 2004

I. Introduction of the Field of Genetics (page numbers and figure numbers refer to your text M. G. A. 2002

Applications of genetics in Agriculture include development of classical plant and animal breeding strategies based on Mendelian genetics, production of transgenic organisms (such as herbicide resistant plants), "pharming", plus others. Recombinant DNA technology has lead to the cloning of medically important genes such as the human insulin gene and human pituitary growth hormone gene so that their products can be easily produced in bacteria and isolated for treatment of human disease. DNA finger printing is useful in all areas of the forensic sciences as well as other areas requiring identification of individuals. Genetics forms one of the foundation disciplines of cancer biology, immunology, cell biology, ecology, evolutionary biology and other areas of biology. Genetics is organized into the following fields of study: Molecular genetics (gene structure and function), Transmission genetics (e.g., Mendelian genetics and quantitative inheritance), Gene expression in the cell and during development, and Population genetics and Evolutionary biology.

The concept of the gene has evolved since Mendel first reported (in 1865) the particulate nature of inheritance. Johannsen in 1909 coined the term gene for Mendel’s particles, and Bateson applied Mendel’s model to polygenic traits such as human stature. Based on his medical studies of patients with metabolic disorders such as galactosemia, Garrod in 1902-04 suggested that the gene produces an enzyme. Subsequently Beadle and Tatum were awarded the Nobel Prize for their work in the 1940’s on Neurospora that demonstrated the one gene – one enzyme relationship. In 1902 Sutton and Boveri proposed the chromosomal basis of inheritance—as cytologists, they recognized that chromosomes in meiosis and fertilization followed the same patterns as Mendel’s particles and, therefore, these particle must be located on the chromosomes. Thomas H. Morgan, beginning in 1909 with Nobel Prize-winning work in Drosophila, demonstrated experimentally that genes are in fact located in a linear order on the chromosomes. Morgan’s student Herman Muller was the first to show that genes (in Drosophila) could be induced to mutate (by X-ray trteatment). This work, done during the 1920’s in Texas, won him the Nobel Prize. In the 1940’s Avery et al demonstrated their transformation studies in bacteria that DNA is the likely genetic molecule. In 1953 Watson and Crick reported their double helical structure of the DNA molecule (awarded Nobel Prize for this work), and suggested in a general way how the molecule might replicate itself and carry information in the sequence of its nucleotides. In the 1960’s Yanofsky and coworkers demonstrated in E. coli that mutant sites in the gene are co-linear with amino acid positions in the polypeptide product of the gene. This was the first strong evidence that information in the gene is translated into amino acid sequence in the gene’s product. In the 50’s and early 60’s, Jacob and Monod in their Nobel Prize-wining work in France determined in E. coli that the gene’s initial product is mRNA which is translated into polypetide and protein structure. Marshall Nirenberg’s Nobel Prize-wining work in the early 60’s with artificial mRNA’s led to the cracking of the genetic code. Today we know that the flow of information is as follows: (gene) DNAà mRNA à polypeptide à functional protein à expression in cell metabolism and differentiation à phenotypic expression.

Mendel’s Model: Genes in pairs, one from each parent, segregate (in Aa) to produce two equally frequent reproductive cells (A) and (a). Random fertilization then results in predictable genetic ratios in F₂ generations- e.g., 3:1. Mendel self-fertilized plants from F2 seeds and observed that from F2 dominant plants 2/3 resulted in 3:1 ratios and 1/3 bred true. Whereas the F2 recessive plants produced only recessive F3 plants. These results demonstrated that the distribution of genotypes in the F2 generation was 1(AA) : 2(Aa) : 1(aa), verifying the segregation of alleles. Mendel performed backcrosses between the F1 plant and both parental plants. He demonstrated that AaBb F1 plants produced four equally frequent types of gametes (AB, Ab, aB, ab) leading to 1:1:1:1 genotypic ratios among the offspring of the backcrosses. Also, different gene pairs (Aa and Bb) assort independently in inheritance to produce dihybrid ratios such as the 9:3:3:1. View Fig. 1-10 for the role of genes in enzyme catalyzed metabolic reactions involved in albinism. Review the Mendel Powerpoint slides on course website.

Be prepared for problems on the exam similar to those on the first homework problem set (autosomal vs sex-linkage, dominance and pedigrees in human genetics, and Mendelian independent assortment). Study the handout of Mendel's F2 data for the seed shape and seed color traits, and be able to use the tree diagram to demonstrate the predicted outcome for his data and other independent pairs of genes.

II. DNA studies and DNA structure Chapter 2 in MGA 2002

In a historical context, in 1866 E. Haeckel proposed that the cell nucleus probably contains hereditary material. Not aware of its function, Frederick Miescher in the 1860’s isolated DNA (he termed it nuclein) from pus and salmon sperm cells, identifying its chemical content as N, C, H, O, and P. This phosphorous-containing compound was distinctly different from protein, a sulfur containing molecule. In 1928 Griffith demonstrated the transformation event in the bacterial pneumonia organism- that some substance from heat-killed virulent (smooth) cells was able to transform non-virulent (rough) cells to the virulent (smooth) cell type. Avery et al 1944 identified the transforming molecule as DNA, having ruled out , through extract digestions, RNA, protein, and polysaccharide capsular material (p. 30, and see website transparencies on transformation studies). In 1952 Hershey and Chase provided additional evidence in the T2 bacteriophage of E. coli that DNA and not protein acts as the genetic material in the bacteriophage. They used the radioactive isotopes ³²P and ³⁵S to label DNA and Protein, respectively, in separate experiments, and followed these isotopes in the life cycle of the bacteriophage (see website transparency). In the 50’s Frankel-Conrat demonstrated that in TMV (tobacco mosaic virus) RNA, not protein, is the genetic material. Review his experiment.

Nucleotide structure (p.27) => deoxyribose sugar (containing 3’ OH group) + N base(purine or pyrimidine) + Phosphate group. The Purines include Adenine and Guanine, double ring compounds (PAG) . The Pyrmidines include Cytosine, Uracil (in RNA) and Thymine, single ring compounds (Py-CUT). Know ribose, deoxy ribose, and dideoxy ribose sugar structures. Chargaff’s rule: [A] = [T] and [G] = [C], but [A + T]/[G + C] ratio is species specific. Why?
Based on Chargaff’s data and x-ray crystallography data from Maurice Wilkins and Rosalind Franklin, Watson and Crick in 1953 published the 3-D structure of DNA (see their article and structural formula for DNA on in-class handout and Fig. 2-3, 2-4, 2-5). Complementarity is a significant feature of double stranded DNA and is the basis of both DNA replication and the transcription process. W/C proposed the semi-conservative model of DNA replication à ====== à ---------- + ----------. They also stated that "the sequence of bases on a single chain does not appear to be restricted in any way". What is the significance of this statement?
The length of one complete turn of the W/C DNA molecule, which includes 10 nucleotide pairs, is 34 Angstroms (1 A = 1 X 10^-10 Meter). If one haploid set of human chromosomes contains 3 X 10⁹ nucleotide pairs of DNA, what is the total length of this DNA if all of the DNA segments were laid end-to-end ? How many inividual segments would there be?
See page 36 for genome sizes of various organisms. Why do some plants and amphibians have 10-100 times more DNA than man? The human genome contains 1% of nucleotides in exons, 25% in introns and 74% in intergenic regions, with approximately 35,000 genes.For comparison of gene content with other organisms, see Table 2-2.
Eukaryotic genes are split into exons (expressed regions) and introns (intervening sequences). See Fig 2-6 for prokaryote vs eukaryote gene structure. Also see Fig 2-7 for the distribution of the numbers of exons in split genes of various organisms. The number of exons/gene is highest in mammals, intermediate in Drosophila and almost absent in the yeast Saccharomyces.
In interphase cells, DNA in chromatin is wrapped around an octamer of histones (basic proteins) at regular intervals to form nucleosomes- see Fig 2-22.
Comparative genomics shows that the gene and chromosomes of closely related organisms are very similar, while more distantly related species show less similarity. See man, cat, and mouse X chromosome (Fig. 2-29).

III. Gene Function: the RNA molecules, Transcription, RNA processing, translation Chapter 3 in MGA 2002

AAD Introductory notes prior to Chapter 3
The human genome (contained in a haploid set of 23 chromosomes) consists of ca 3 billion nucleotide pairs. The total length of the human genome is 3.4 angstroms/nucleotide pair X 3billion nucleotide pairs = 3.4 X10^-10 Meters X 3 X 10⁹= ca 1 Meter. (1Angstrom = 1 X 10^-10 Meters)
The human genome contains 30,000-40,000 genes; most of these are split into the coding regions (exons) and non-coding intervening sequences (introns). (Use the back button on your browser to link to the two articles (in NATURE and SCIENCE) which report the final summaries of the human genome project studies.) These genes are made up of unique sequence DNA. A large percentage of the genome is made up of repetitive sequence DNA, which consists of repeats of short to long nucleotide sequences that do not code for proteins. In summary, it is estimated that only approximately 1% of of the 3 billion nucleotide pairs in the human genome are coding for amino acid sequence (codons) or the various RNA sequences(rRNA, tRNA, snRNA). The remainder of the DNA contains non-coding introns (25%) and intergenic DNA (74%).

During transcription of a gene, one strand of DNA (the anti sense strand or template strand) serves as a template on which is assembled a complementary strand of RNA under the action of RNA polymerase. Three major types of RNA are identifiable: messenger RNA (product of protein coding genes), ribosomal RNA (from rRNA genes), and transfer RNA (from tRNA genes), plus a fourth class known as snRNA (small nuclear RNA instrumental in RNA processing in eukaryotes). Review the abstract and summary section of the article handed out in class in class “Life with 6000 Genes”, for the numbers of the different types of genes in yeast as a model eukaryotic organism. For example, in the yeast genome, there are 275 tRNA genes but they occur in 43 families, indicating that each tRNA gene is repeated on average six times, usually in tandem repeats.

Transcription is initiated at the promoter region of a gene where RNA polymerase binds and then continues on to the termination signal sequence at the end of the gene (Fig. 3.2, 3.4, 3.5). The different RNA polymerases (p. 62) catalyze the formation of RNA from 5’ to 3’direction as follows:

DNA section being transcribed:

5’ end …...A T T C G C A A T C G G T G A A …. sense strand

3’ end ……T A A G C G T T A G C C A C T T ……..anti sense or template strand

¦
RNA

5’ end ……A U U C G C A A à ….. 3’ end

The promoter regions of genes have highly conserved nucleotide sequences that serve as recognition sites for the binding of RNA polymerase. The term conserved sequence refers to the fact that mutations in these regions are usually detrimental to the organism and are eliminated through natural selection; therefore, most promoters in most organsims have nearly the same such sequence because they all respond to the same RNA polymerase molecule. In prokaryotes there are two conserved sequences, one at –10 and the second at –35 nucleotides upstream from the beginning of transcription at nucleotide position 0. See fig 3-9, which shows the promoters of 13 different E. coli genes. They all share nearly the same nucleotide sequences at postions -10 and -35. These sequences are highly conserved in evolution- mutations in these regions are eliminated through selection.
In eukaryotes the conserved TATA box is at position –25 and the conserved CAAT box is at –75. Review in detail the mutation studies of the mouse beta globin promoter used to identify important conserved sequences in this promoter on the course website transparency.
The initiation of transcription in prokaryotes is dependent on the binding of the multi subunit RNA polymerase to the promoter. The sigma factor of RNA polymerase is the important subunit in this recognition event. In eukaryotes, a large number of transcription factors (proteins) plus one of the three RNA polymerases bind to the promoter to initiate transcription. A second important region, the enhancer, is present near most eukaryotic genes to facilitate initiation of transcription. Elongation of the RNA transcript proceeds under the action of RNA polymerase and several other proteins. Termination of transcription is signaled by conserved termination sequences at the end of the gene. In Prokaryotes two termination sequences have been identified: intrinsic terminator and rho dependent terminator. In eukaryotes, termination is more varied and complex. Several genes contain a highly conserved sequence AAUAAA in mRNA downstream from which cleavage of the RNA molecule occurs, thus ending transcription of this gene (seeFig 3-11).
Ribosomal RNA genes are clustered and are transcribed as a unit. Specifically, in prokaryotes, the 7s, 16s and 23s subunits are adjacent to each other and occur in seven tandem repeats. In eukaryotes, the 5.8s, 16s and 28s rRNA subunits occur in repeating clusters of up to 1000 copies on a chromosome. This chromosomal region is termed the nucleolar organizer region (NOR) and gives rise to the nucleolus (see Fig 2-16). It is inside the nucleolus where rRNA is being synthesized and processed and where ribosomal subunits are assembled (see Fig. 3-6 and 3-23).
In prokaryotes, transcription and translation are coupled so that as mRNA is forming, ribosomes immediately attach and begin translating the mRNA message into polypeptides (see the classic electronmicrograph of these events on the course website transparencies). In eukaryotes, transcription occurs within the nucleus and translation occurs on riboosmes in the cytoplasm, outside of the nucleus.
After the discovery of methods to sequence genomic DNA in the 1970’s , it became apparent that certain nucleotides sequences in the eukaryotic gene were not represented by corresponding sequences of amino acids in the gene’s protein product. The eukaryotic gene is split into alternating exon sequences (that are expressed in amino acid sequences) and introns, intervening sequences that are not expressed. Due to this split nature of the eukaryotic gene, the initial transcript of a protein coding gene (the pre mRNA) must be processed to remove the introns. Intron removal and exon splicing occur under the action of the splicesome, a complex unit that includes small nuclear RNA molecules (snRNA) in combination with proteins to make a snRNP (small nuclear ribonucleoprotein) that recognizes the junction sequences between an exon and its adjacent intron. Most introns follow the GT-AG rule, beginning at the 5’ end with G T and ending at the 3’ end in AG. Once removed, the introns are degraded and play no further coding role. Study Figs 3-12, 13, 14, 15 for details of exon splicing, and pay particular attention to the highly conserved sequences at exon/intron junction. What is the result of mutation at this junction? Two additional events occur in the processing of the pre mRNA: a poly A tail is added to the 3’ end and a special Guanine cap is formed at the 5’ end. Study Fig. 3-16 for overview of these events. The Guanine cap serves to prevent degradation of the mRNA and also appears to serve as the site where the ribosome binds to the mRNA for translation. The poly a tail at the 3’ end of mRNA includes from 100 – 300 adenine ribonucleotides that are enzymatically added after the initial RNA molecule has been cleaved. This tail serves to prevent degradation.

Structure of the eukaryotic split gene ( and see fig 3-16)

Human and chimp beta globin genes have been sequenced and compared. They contain identical nucleotide sequences in their exons and therefore identical amino acid sequences. There a slight differences in their introns.

What is the exon shuffling model of eukaryotic gene evolution- proposed by Walter Gilbert?

Translation

DNA à mRNA à Polypeptide à functional protein (enzymes, cell receptors, antibodies, etc).

Amino acid structural formula, R groups and peptide bonding (see Fig. 3-17, 3-18). Be able to draw the structural formula of a tri peptide. Yanofsky demonstrated colinearity of a gene and its protein (read p. 78). [Frame shift mutants were used by Crick to demonstrate the triplet nature of genetic code – the addition (or deletion) of one or two nucleotides in a gene caused a mutant phenotype, whereas the addition of three nucleotides corrected the reading frame and produced a nearly wildtype phenotype (reversion to wildtype).] [this info not on first exam S 2003)]

The genetic code (Fig. 3-20) is a triplet code, universal, degenerate, comma-less, non-overlapping, with start/stop codons. Degeneracy due to multiple (2 or 3) t-RNA’s for the same amino acid and third letter "wobble" between codon and anti-codon (see Table 3.2).
[To crack the genetic code, Nirenberg (1961) synthesized artificial mRNA molecules and translated these in an in-vitro system to assign amino acids to the codons. For example, poly U => phe-phe-phe… This was followed by the use of triplet binding assay (triplet mRNA codon + ribosome binds a specific tRNA-amino acid) which permitted assignment of most of the 64 codons to their amino acid] [This info not on first exam S 2003.]
Universal start codon is AUG which specifies methionine, and the three stop codons are UAG, UAA, and UGA. In eukaryotes, AUG in the start or initiation position is recognized by Met-tRNA(i) and AUG codons located internally in the gene are recognized by a second type of tRNA (Met-tRNAm). The first amino acid in a polypeptide (at its NH2 end) is methionine, but this amino acid is often removed in post translational processing of the polypeptide.

Roles of RNA’s in translation. Ribosomal RNA’s combine with proteins to form large and small subunits of the ribosome which bind to mRNA and lead to translation (see Fig. 3-23). Transfer RNA’s, approximately 45 different species in number, bind to amino acids and deliver them to ribosome for translation. The charging reactions result in the bonding of an amino acid to its tRNA and are catalyzed by 20 unique enzymes, the aminoacyl-tRNA synthetases (study transparency on website, to be added 2-7-03). The anti-codon of tRNA pairs (with third base wobble) on the ribosome with its complementary codon in mRNA and delivers its amino acid, through formation of the peptide bond, to the growing polypeptide chain. See Fig. 3-21, 3-22 for tRNA structure and codon-anticodon pairing.

Translation. The initiation complex includes tRNA-fmet (in bacteria), 5’ end of mRNA, small and large subunits of ribosome and several protein initiation factors (see website trasparencies on translation in prokaryotes). The fmet-tRNA first occupies the and then moves into the P site on the ribosome, leaving the A site open to receive the next (second) amino adid-tRNA molecule. For similarities and differences in initiation of translation in Eukaryotes vs prokaryotes, see website transparencies.. During the elongation process, the enzyme peptidyl transferase catalyzes the peptide bond between the two amino acids at the P and A sites in the ribosomes. This frees the tRNA in the P site from its amino acid, and the ribosome then moves over three nucleotides on the mRNA . The A site is now open for the next tRNA-amino acid to come into position. The high energy molecule GTP provides the energy for this translocation process. Termination of translation is signaled by the stop codon in mRNA. A releasing factor (RF) catalyzes termination when a stop codon occupies the A site, thus terminating translation and releasing the newly made polypeptide, the mRNA and the ribosomal subunits. See websote transparencies.

Sickle cell anemia is due to a single amino acid substitution in the beta globin polypeptide (glu, a negatively charded aa is replaced by valine, an uncharged aa, at positopn #6). Pauling in the 1950s showed that this mutation changes the charge of the Hgb molecule (see website transparency). Pauling's study was the first to show a chemical change in a protein as a result of mutation in a gene.

Yanofsky demonstrated the colinear relationship between mutant sites in the tryp synthetase gene in E. coli and amino acid substitutions in the resulting proteins. This demonstration of colinearity provided strong evidence that the role of the gene is to determine amino sequence in the polypeptide product of the gene. See details of his study on p. 78.

Examine the handout of metabolic pathways, and note the genetic disorders associated with this system. Explain the two consequences of a metabolic block; 1) lack of end product, as in albinism, 2) accumulation of precursor molecule as in PKU. Two albino parents have all normally pigmented children. Explain, with assigned genotypes, using the information for albinism on the handout.

Beadle and Tatum received the Nobel prize for demonstrating the one gene – one enzyme model in Neurospora (see details of their study of nutritional or auxotrophic mutants on the hand out). They did "single cell genetics" with the haploid Neurospora. How were they able to use minimal and complete media to identify the specific nutritional requirement of their mutants? How can metabolic mutants be used to determine the steps in the metabolic pathway for the synthesis of an amino acid?(see handout and p. 76 for details of their study). Today, we state that one gene codes for one polypeptide- why?

Archibald Garrod reported on the likely role of genes-enzymes in metabolic disorders such as alkaptonuria and albinism in his 1902 book Inborn Errors of Metabolism. He was the first to suggest that genes control the production of enzymes (ferments), and that these traits are inherited in huiman pedigrees according to Mendelian principles. He fed Alkaptonuria patients large quantities of homogentisic acid which they excreted in their urine which turned black when exposed to air. Garrod concluded that these patients lacked an enzyme that normally degrades homogentisic acid .

Posttranslational modification of proteins involves various kinds of changes to the polypeptide that must occur prior to its proper functioning. Chaperones (proteins) often assist a polypeptide to fold into its final three dimensional form.
Protein levels of structure: Primary (a.a. sequence), Secondary (alpha helix and beta pleated sheet) structure maintained by H bonding, Tertiary (3-D), and Quarternary (multiple polypeptides make up the final functional protein, e.g., hemoglobin = two alpha globin chains + two beta globin chains). See Fig 3-18

BSE (bovine spongioform encephalopathy) or mad cow disease is caused by a prion that acts to cause the conversion of alpha helix protein structure to beta pleated sheet form. This disease has been transmitted to man by eating infected beef and is identified as a new variant of the brain-wasting Creutzfeldt-Jacob disease (vCJD). The infectious agent in this case is a brain protein, the prion. (see BSE article in the journal SCIENCE )