BIOL 3300 - Exam 3 - Dimuth Siritunga PDF

Preview

Summary

Dimuth Siritunga...

Description

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

Linkage and Recombination Chapter 7: pp 120 – 131 Linked genes tend to be inherited together because they are located on the same chromosome:  Each chromosome has hundreds or thousands of genes. (Genes may not be on different chromosomes, no linkage)  Genes located on the same chromosome, linked genes, tend to be inherited together because the chromosome is passed along as a unit.  Results of crosses with linked genes deviate from those expected according to independent assortment.  Linked genes: (genes located on the same chromosome) move together through meiosis and fertilization. o Under normal Mendelian Genetics, we would not expect linked genes to recombine into assortments of alleles not found in the parents.  Morgan reasoned that body color and wing shape are usually inherited together because their genes are on the same chromosome.  The other two phenotypes (gray-vestigial and black-normal) were fewer than expected (and totally unexpected from dependent assortment)  These new phenotypic variations must be the result of crossing over.  The results of Morgan’s testcross for body color and wing shape did not confirm independent assortment or compete linkage. o Independent assortment: testcross = 1:1:1:1 phenotypic ratio o Completely linked testcross = 1:1:0:0 with only parental phenotype showing Recombinant mapping with a three-point cross:  Cross 1 & 2, Cross 2 & 3 and Cross 1 & 3  Units: Centimorgans or Milimorgans  The farther apart two genes are, the higher the probability that a crossover will occur between them and therefore a higher recombination frequency. o The greater the distance between two genes, the more points between them where crossing over can occur.  Alfred Sturtevant (Morgan’s Student) used recombination frequencies from fruit fly crosses to map the relative position of genes along chromosomes, a linkage map. Linkage Maps:  Maximum distance in linkage maps is 50%  Tells you if two genes are in the same chromosome, and how close they are.  Were used in agriculture for a long time.  If the two low side are next to each other, good genes may not be inherited by progeny. o Mother: Good – Bad o Father: Bad – Good  The map of relative position of three fruit fly genes, body color (b), wing size (vg) and eye color (cn). o 9% (b-cn) + 9.5% (cn-vg) > 17% (b-vg)  This results from multiple crossing over events:

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

o A second crossing over “cancels out” the first and reduces the observed number of recombinant offspring. o Genes farther apart (For example, b – vg) are more likely to experience multiple crossing over events. (in multiple crossing over events, the parental number is higher than the recombinant number, because the second event cancels the first recombination.)

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

Regulation in Prokaryotes Chapter 8 & Chapter 15      

Viruses and bacteria are the simplest biological systems (single-celled organisms) Microbiologists provided most of the evidence that genes are made of DNA and they worked out most of the major steps in DNA replication, transcription and translation. Viruses and bacteria also have interesting, unique genetic features with implications for understanding diseases that they cause. (either to humans or other food supplies, and as a result, they are constantly studied) Bacteria are prokaryotic organisms. (A virus is also considered a prokaryote) Their cells are much smaller and more simply organized than those of eukaryotes, such as plants and animals. Viruses are smaller and simpler still, lacking the structure and most metabolic machinery in cells. Most viruses are little more than aggregates of nucleic acids and protein (genes in a protein coat) o DNA virus: genetic information is from DNA o RNA virus: genetic information is from RNA

Genetics of bacteria:  Bacteria are very adaptable.  This is true in the evolutionary sense of adaptation via natural selection and the physiological sense of adjustment to changes in the environment by individual bacteria. (They adapt by changing their genes, because they have very rapid reproduction rates)  The major component of the bacterial genome is one double-stranded, circular DNA molecule. o For E. coli, the chromosomal DNA consists of about 4.6 million nucleotide pairs with about 4,300 genes. (humans have billions of nucleotides) o This is 100 times more DNA than in typical virus and 1000 times less than in a typical eukaryote cell. o Tight coiling of the DNA results in a dense region of DNA, called the nucleoid, not bounded by a membrane.  In addition, many bacteria have plasmids, much smaller circles of DNA. o Each plasmid has only a small number of genes, from just a few to several dozen.  Bacterial cells divide by binary fission. o The first opening that occurs in the circular DNA is in the Ori (Origin of Replication) where the hydrogen bonds are being broken in two directions. Then DNA polymerase comes and makes new DNA. (One becomes Two) o E. coli will multiply in under 20 minutes.  Bacteria proliferates very rapidly in a favorable natural or laboratory environment. o Under optimal laboratory conditions E. coli can divide every 20 minutes, producing a colony of 107 to 108 bacteria in as little as 12 hours.  Through binary fission, most of the bacteria in a colony are genetically identical to the parent cell. o However, the spontaneous mutation rate of E. coli is 1 x 10-7 mutations per gene per cell division. (Every time binary fission occurs; the children have 4.6 mutations different than those that survived)

BIOL 3300



 

Prof. Dimuth Siritunga

Partial Exam #2

o This will produce about 2000 bacteria in the human colon that have a mutation in that gene per day. Bacteria has a fast-reproductive rate (20-30 minutes), and a slow mutation rate (4.6). They produce new genetic variation by mutations. New mutations, though individually rare, can have a significant impact on genetic diversity when reproductive rates are very high because of short generation spans. Individual bacteria that are genetically well equipped for the local environment clone themselves more prolifically than do less fit individuals. In contrast, organisms with slower reproduction rates (like humans) create most genetic variation not by novel alleles produced through mutation, but by sexual recombination of existing alleles.

Genetic recombination produces new bacterial strains:  In addition to mutations, genetic recombination also generates diversity within bacterial populations.  Here, recombination is defined as the combining of DNA from two individuals into a single genome.  Recombination occurs through three processes: o Transduction o Transformation o Conjugation  The impact of recombination can be observed when two mutant E. coli strains are combined. Bacteria doesn’t rely on mutations; it has a system of recombination of DNA. o Mutation (-) where you can’t make the amino acid o Wild type (+) you can make the amino acid o Minimal medium (only contains water and sugar): bacteria die o Minimal medium with amino acids that the bacteria can’t produce: bacteria survives (colony is wild type for all amino acids, and has whole new genes) Transformation; alteration of a bacterial cell’s genotype by the uptake of naked (not inside another bacteria; usually from dead bacteria), foreign DNA from the surrounding environment.  For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells.  This occurs when a live nonpathogenic cell takes up a piece of DNA that happens to include the allele for pathogenicity from dead, broken-open bacteria.  Many bacterial species have surface proteins that are specialized for the uptake of naked DNA. o These proteins recognize and transport only DNA from closely related bacterial species. o While E. coli lacks these specialized mechanisms, it can be induced to take up small pieces of DNA if cultured in a medium with a relatively high concentration of calcium ions.

BIOL 3300



Prof. Dimuth Siritunga

Partial Exam #2

o In biotechnology, this technique has been used to introduce foreign DNA into E.coli. (Also, by giving it heat-shock, the cell wall opens up enough, allowing DNA to go in). Diagram: o Bacterial cell and bacteria plasmid (goes through binary fission). At some point, a signal to look for DNA makes it find DNA. It requires the Competent Bacterium Receptor Site (Surface Protein) and the foreign DNA is taken in. o The foreign DNA may be destroyed, or integrated into the bacteria’s genome. The double strand plasmid’s outer DNA and it becomes two: one different from original (contains foreign DNA) and one is the same as the original.

Transduction:  Occurs when a phage carries bacterial genes from one host cell to another.  Diagram: o DNA Phage has attached to host chromosome and inserts its genome and has multiplied it. Then, it destroys the bacterial plasmid, which is cut into small pieces. Now the cell is a mixture of pieces of phage and pieces of original plasmid. When the viral coat protein is made, it makes mistakes as it encapsulates bacterial genome. Mature phages then contain a random piece of bacteria genome. Then when this cycle is repeated, some bacteria cells will incorporate the phagecarried bacteria genome, resulting in new bacterial genome. Conjugation:  Transfers genetic material between two bacterial cells that are temporarily joined.  A sex pilus from the male initially joins the two cells and creates a cytoplasmic bridge between cells. (temporary cytoplasmic connection between one bacteria and another, the ability of producing sex pilus is a result of maleness)  “Maleness”, the ability to form a sex pilus and donate DNA, results from an F-factor as a section of the bacterial chromosome or as a plasmid. (F-factor = male). o When the male sees a female, which doesn’t contain F-factor, and the factor’s genes are trigged, making proteins that enable the sex pilus.  They usually have only a few genes that are not required for normal survival and reproduction. o Plasmid genes are advantageous in stressful condition.  The F plasmid facilitates genetic recombination when environmental conditions no longer favor existing strains.  The fertility factor or its F plasmid consists of about 25 genes, most required for the production of the sex pili.  Diagram: o Two bacteria cells (F+ and F-), the cytoplasmic bridge and the F+ gives the F- a copy of its F factor, making it then an F+. o NO GENETIC RECOMBINATION IN THIS PROCESS  The plasmid form of F factor can become integrated into the bacterial chromosome.  The resulting Hfr cell (high frequency recombination) functions as a male during conjugation. o Hfr + F-: it makes the sex pilis, and it tries to give a copy of the F factor.

BIOL 3300 

Prof. Dimuth Siritunga

Partial Exam #2

Diagram 2: o Conversion of F- to an Hfr state occurs by integrating the F factor into the bacterial chromosome. o The point of integration determines the origin (O) of transfer. o During conjugation, an enzyme nicks the F factor now integrated into the host chromosome, initiating transfer of the chromosome at that point. o Conjugation is usually interrupted prior to complete transfer.  F- may become attacked by Hfr = change in its genome  F+ stops being attacked by Hfr

In all three methods, the result is a completely new bacteria with a new genome. This is how bacteria changes its DNA in order to adapt to the environment. Metabolic Control  The control of gene expression enables individual bacteria to adjust their metabolism to environmental change  An individual bacterium, locked into the genome that it has inherited, can cope with environmental fluctuations by exerting metabolic control. o Quantitative: cells vary the number of specific enzyme molecules by regulating gene expression. o Qualitative: cells adjust the activity/ability of enzymes already present (for example, by feedback inhibition)  Precursor (compound) ------ Enzyme 1-5 -------- Tryptophan (Product) o Every enzyme has a gene that codes for it. o Feedback Mechanisms: the end product can bind the DNA of any gene, stopping transcription. This lowers the production of the valuable product. o Another stops at the beginning by placing the valuable product in place of the precursor. (Shuts down whole pathway)  For example, the tryptophan biosynthesis pathway demonstrates both levels of control. (Post-Translational) o Qualitative: if tryptophan levels are high, some of the tryptophan molecules can inhibit the first enzyme in the pathway. The regulatory mechanisms with the largest effects on phenotype act at the level of transcription (prokaryotic and/or eukaryotic): - Occurs during Transcription, processing of RNA, Translation or Post-Translational. Operons: - In 1961, Francois Jacob and Jacques Monod proposed the operon model for the control of gene expression in bacteria. - Has three main domains: o Domain 1 - Structural Genes: all one after the other o Domain 2 - Regulatory Region: what controls genes and where the promoter and operator sequence are found. o Domain 3 – Repressor Domain: one specific gene (usually only one protein does the join)

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

By itself, an operon is ON and RNA polymerase can bond to the promoter and transcribe the genes. Repressible operon: Tryptophan Operon (Always ON) - Tryptophan: Critical amino acid required by prokaryotes. Meaning it is always on. - Genes: A, B, C, D, E, code for five different proteins (enzymes) that are controlled by a regulatory area. - Regulatory area: o Promoter Sequence / Region: attracts RNA polymerase to do transcription. (ACTIVE TRANSCRIPTION) o Operator Sequence / Region: has the ability to bind repressor proteins. The RNA polymerase will not be able to move. (INACTIVE TRANSCRIPTION) - Under normal circumstances, the tryptophan repressor is inactive. - If the repressor is active, the rate of transcription goes down. Thus no new tryptophan will be made. -

If a repressor protein, a product of a regulatory gene, binds to the operator, it can prevent transcription. o Each repressor protein recognizes and binds only to the operator of a certain operon.

-

Binding by the repressor to the operator is reversible. The trp operon is an example of a repressible operon, one that is inhibited when a specific small molecule binds allosterically to a regulatory protein In contrast, an inducible operon is stimulated when a specific small molecule interacts with a regulatory protein. o The regulatory protein is active (inhibitory) as synthesized, and the operon is off. o Allosteric binding by an inducer molecule makes the regulatory protein inactive.

-

The lac operon contains a series of genes that code for enzymes that play a major role in the hydrolysis and metabolism of lactose. - Under normal circumstances, the lac repressor is active, and the operon is always off. - The repressor molecule is bound and active, thus the RNA polymerase cannot do any work. (All three genes have no production of enzymes – Transcription is shut down) - To inactivate the lac repressor, lactose is required in order to stop the repressor molecule from binding and blocking the RNA polymerase. Inducible operon: Lactose Operon (Always OFF) (It is like a dimmer) - Operon that is on and must be induced. - Under normal circumstances, the lac repressor is active. + : means no mutations in that gene - : means there is a mutation in that gene, and the functional lac “gene” protein will not be made

BIOL 3300 -

-

-

Prof. Dimuth Siritunga

Partial Exam #2

Repressible enzymes generally function in anabolic (formation) pathways, synthesizing end products. o When the end product is present in sufficient quantities, the cell can allocate its resources to other uses. Inducible enzymes usually function in catabolic (breakdown) pathways, digesting nutrients to simpler molecules. o By producing the appropriate enzymes only when the nutrient is available, the cell avoids making proteins that have nothing to do. Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates. o If glucose levels are low (along with energy levels), then cyclic AMP (cAMP) binds to cAMP receptor protein (CRP) which activates transcription.  AMP: adenosine monophosphate  ATP and AMP are inversely proportional.  CRP: can make transcription of the lac operon occur faster o It regulates the consumption or breakdown of lactose by checking its energy levels.

Explanation: Operon must be on by the presence of lactose, but then the level of need is determined. If levels are low, ATP is low, and AMP is high. -

Overall energy levels in the cell determine the level of transcription, a “volume” control, through CRP. CRP works on several operons that encode enzymes used in catabolic pathways. o If glucose is present and CRP is inactive, then the synthesis of enzymes that catabolize other compounds is slowed. o If glucose levels are low and CRP is active, then the genes which produce enzymes that catabolize whichever other fuel is present will be transcribed at high levels.

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

Chapter 15: Gene Regulation in Eukaryotes The organization and control of eukaryotic genomes -

-

-

-

Gene expression in eukaryotes has two main differences from the same process in prokaryotes. o First, the typical multicellular eukaryotic genome is much larger than that of a bacterium. o Second, cell specialization limits the expression of many genes to specific cells.  Bacteria have no cell specialization, as each cell type has the same genome, but only a sub-set of it (genes are silenced depending on their location’s need) There are an estimated 20,000 genes in the human genome that includes an enormous amount of DNA that does not program the synthesis of RNA or protein. o 97% of your DNA does not synthesize RNA or proteins. o We are only about 3% of coding DNA This DNA is elaborately organized. o Not only is the DNA associated with protein to from chromatin, but the chromatin is organized into higher organization levels. Level of packing is one way that gene expression is regulated. o Densely packed areas are inactivated o Loosely packed areas are being actively transcribed (RNA polymerase may do transcription)

What alters tightly or loosely packed DNA? - A tightly packed DNA will have a slower rate of transcription as opposed to a loosely packed DNA. - Histone proteins are responsible for the first level of DNA packaging. o Their positively charged amino acids bind tightly to negatively charged DNA (due to charge differences) o The five types of histones are very similar from one eukaryote to another and are even present in bacteria. - Unfolded chromatin has the appearance of beads on a string, a nucleosome, in which DNA winds around a core of histone proteins. - In a mitotic chromosome, the looped domains coil and fold to produce the characteristic metaphase chromosome. - These packing steps are highly specific and precise with particular genes located in the same place. - The binding between DNA and histones is not permanent, as when the cell requires it, it may loosen its grip to the histones and become available for transcription.

BIOL 3300

Prof. Dimuth Siritunga

Partial Exam #2

Each cell of a multicellular eukaryote expresses only a small fraction of its genes: - Like unicellular organisms, the tens of thousands of genes in the cells of multicellular eukaryotes are continually t...