BIO 345 Introductory Genetics     
Review Items for First Exam       Spring 2002                 posted 2-1-02

I. Introduction of the Field of Genetics (Chapter 1 Fairbanks/Anderson plus lecture notes)

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. 1.9 and 1.11 for the role of genes in enzyme catalyzed metabolic reactions involved in flower color.    Review the Mendel Powerpoint slides.

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.  Extra review Mendelian genetics problems are available here.

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 (Fig. 2.2). Avery et al 1944 identified the transforming molecule as DNA, having ruled out , through extract digestions,   RNA, protein, and polysaccharide capsular material (Fig. 2.3). 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 (Fig. 2.4). 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. 26, 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 (Example 2.2). 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. 2.10, 2.11). 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?

Models of DNA replication : Semi-conservative, conservative and dispersive (Fig 2.13). Meselson and Stahl (1958) used the isotopes ¹⁴N and N¹⁵ to label newly synthesized and old DNA chains.  Through the use of density gradient centrifugation they  demonstrated that DNA replication in E. coli follows the semi-conservative model (see Fig. 2.16). Be prepared to describe th;is study in detail.   Taylor et al showed in bean root tips that DNA replication in the mitotic cell cycle also follows the semi-conservative model. They used the radioactive isotope of H (tritium) to label thymidine in newly synthesized DNA and detected this label through autoradiography (exposure through radioactive decay of photographic emulsion spread over a metaphase chromosome spread).

Arthur Kornberg (1950’s) isolated the DNA replication enzyme, DNA-directed DNA polymerase (now Pol I), in E. coli.He demonstrated that the following in vitro reaction took place:

  Template DNA (e.g., from cow) + dNTPs(all four deoxyribonucleoside triphosphates where N=A, G, C, and T)  + Polymerase + Mg ions   ------>   newly synthesized cow DNA  + p-p (inroganic phosphate)

He later received the Nobel Prize for this work. Soon after his discovery, mutant strains were identified that lacked his enzyme and had a very high mutation rate. Three DNA polymerases have been identified in bacteria – Pol I, II, and III. All catalyze the synthesis of DNA in the 5’ to 3’ direction, using a double stranded DNA substrate. Also, all three have a profreading function and a 3' to 5' exonuclease function to remove mismatched nucleotides.   (See Fig. 2.23 for proofreading functions of the DNA polymerases). The POL III enzyme is the major polymerizing enzyme in E. coli. The POL I enzyme has a 3' to 5' exonuclease function and removes the RNA primer during the replication process and then replaces primer with deoxy nucleotides.  See Table 2.1 for the functions of all three enzymes, but note error in this table: DNA polymerase III has a 3’ to 5’ exonuclease function, not a 5’ to 3’ exonuclease direction. Also see Table 2.2 for functions of the five  DNA polymerases in Eukaryotes. 
DNA replication of a double stranded molecule occurs in a semi-discontinuous manner, the lagging chain forming in segments (Okazaki fragments) and the leading chain in a continuous manner(see class handout on this).   Study Figures 2.22 and 2.25 in detail for the models and enzymes involved in DNA replication in Prokaryotes and Eukaryotes. In prokaryotes, primase catalyzes the formation of complementary primer RNA which, with its DNA complementary strand, forms a double stranded substrate upon which the DNA polymerases act to extend a DNA chain in the 5’ to 3’ direction. DNA ligase forms the final phosphodiester bond between two adjacent nucleotides to join two sections of the lagging DNA strand. Origins of replication (ORI) contain short repeating nucleotide sequences (13 mers and 9 mers) which are recognized by DNA binding proteins to open up the DNA molecule so that DNA replication can begin. One origin of replication exists in most bacterial chromosomes, while many origins are found along a eukaryotic chromosome (study Fig. 2.26 and 2.27).

Telomeres are repeated short nucleotide sequences ( TTAGGG in humans) at the ends of eukaryotic chromosomes. The enzyme telomerase produced in germline cells adds these segments and facilitates replication of the ends without the shortening that occurs in somatic cell division. Study Fig.2.33 for details of the telomerase story. Cells in tissue culture have a limited number of doublings due to absence of telomerase activity, whereas some tumor cell lines are immortal in culture due to the expression of telomerase in these tumor cell. A functioning telomerase gene was inserted into somatic cells (see Jan 98 Science article hand out) and was shown to prevent chromosome shortening and thus extended the number of cell divisions in these genetically engineered cells.

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.
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 discussed  in class “Life with 6000 Genes”, for the numbers of the different types of genes in yeast as a model eukaryotic organism. 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 (Table 3.1) 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. In eukaryotes the conserved TATA box is at position –25 and the conserved CAAT box is at –75.  See Figures 3.6 and 3.8 for details of promoter sequences in prokaryotes and eukaryotes.   Also review in detail the mutation studies of the mouse beta globin promoter used to identify important conserved sequences in this promoter (Example 3.2 and see Fig. 3.9). 
The initiation of transcription in prokaryotes is dependent on the binding of the multi subunit RNA polymerase to the promoter.  The sigma factor is the important subunit in this recognition event (see Fig. 3.7).  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 (study Fig. 3.11).  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 (see Fig. 3.13 and 3.14 for details of these systems of termination).  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 (see Fig. 3.15). 
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.   It is inside the nucleolus where rRNA is being synthesized and processed and where  ribosomal subunits are assembled (Fig. 3.16, 3.17). 
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 in Fig. 3.3).  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.  See Fig. 3.21 for details of these events. 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 (Fig. 3.19, 3.20).  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.  

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

Amino acid structural formula, R groups and peptide bonding (see Fig. 4.1, 4.2, 4.3). Be able to draw the structural formula of a tri peptide.  Yanofsky demonstrated colinearity of a gene and its protein (read Example 4.1, p. 92). 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).

The genetic code (Fig. 4.4) 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 4.2). 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 (see p. 106 and Fig. 4.21). 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. 4.8, 4.9, 4.10). Transfer RNA’s, approximately 40 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 Fig. 4.14, 4.15). 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. 4.12 for tRNA structures. Review handout article: Life with 6000 Genes.

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 Fig. 4.22, 4.23). The fmet-tRNA occupies the P site on the ribosome leaving the A site open to receive the next (second) amino-tRNA molecule. For similarities and differences in initiation of translation in Eukaryotes vs prokaryotes, see Fig. 4.24. 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. See Fig. 4.25 for elongation in Prokaryotes. 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 Fig. 4.26.

Posttranslational modification of proteins involves various kinds of changes to the polypeptide that must occur prior to its proper functioning. Chaperones often assist a polypeptide 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).

Structure of the eukaryotic split gene:

5’ end of sense strand….. promoter region | leader region - |ATG (start codon) of exon 1 | GT…intron 1…..AG | exon 2 | GT….intron 2….AG | …… | last exon TGS (one of the stop codons) | trailer region with transcription termination sequence and poly A tail attachment site….. 3’ end. Study handout of beta globin gene structure in the rabbit, and review Figure 6.2 in detal for the components of the human beta globin gene.

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.

What is the exon shuffling model of eukaryotic gene structure?

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).