Exam 4 Genetics

Chapter 6: Genetics of Bacteria and Bacteriophages
Bacteria is easy to grow
Haploid- any mutation is expressed and passed on
==Nutritional mutants==: missing ability to synthesize an organic component (his-)
==Antibiotic mutants==: are resistant to an antibiotic (strR)
==Prototrophs==: can grow on minimal medium; can synthesize all essential organic compounds. Wild-type for all growth requirements.
==Auxotrophs==: have lost, through mutation, the ability to synthesize one or more organic components and must be grown on supplemented medium. Ex: synthesis of histidine. his+=prototroph, his-=auxotroph
==Minimal medium==: nutrient components are very simple, only consisting of an organic C source (e.g., glucose, lactose) and various inorganic ions (Na+, K+,Mg2+,Ca2+, etc.)
==Complete medium==: medium that has been extensively supplemented to contain all the growth requirements of that strain
==Selective media==- limiting the components in the medium. A way to isolate certain genotypes from a mixed population.
Bacterial “sex”= conjugation
Genetic recombination=replacement, not reciprocal exchange
Genetic info from one bacterium is transferred to another. Recombines at independent locations to become wt cells. Prototrophs result from 2 auxotrophs.

The F factor
==F factor===fertility factor
Allows unidirectional exchange of genetic material
==F factor==: a plasmid with genes that function in process of conjugation. Confers ability to donate genetic material thru conjugation. One bacterium is a donor, other is the recipient. donor=F+, recipient=F-. The physical connection between the bacteria=F pilus (sex pilus) Physical contact is required to exchange DNA. Filter between strains A&B -> no recombination -> no prototrophs produced

Process of F+ x F- conjugation (mating)

Copy of F factor is transferred from F+ cell to the F- recipient, converting the recipient to the F+ state. After conjugation, recipient F- cells always become F+.
Some F plasmids spontaneously integrate into the bacterial chromosome. This can lead to recombination after conjugation – but at a very low frequency.
==Hfr bacteria=== high-frequency recombination (a special class of F+ cells)

integration of F plasmid into a bacterial genome
conjugation – bacterial genes are transmitted
transferred genes may recombine with the recipient Conjugation for only a short time – never the entire Hfr chromosome, and rarely the entire F factor. Therefore, the recipient remains F-.

Hfr mating

Transferred DNA usually then integrates into recipient genome
Bacterial gene mapping: interrupted mating
Length of conjugation = how much of donor genome is transmitted.
Order of gene transfer influences # of recombinants
1st gene= most recombinants
2nd gene= next-most, etc.

Protocol:

Incubate mix of Hfr and F- bacteria
periodically take sample, put in blender.
Isolate bacteria and count # recombinants
Order of genes is based on # of recombinants
“Distance” = minutes until a recombinant is detected

Detecting recombinant bacteria: selective plating
Hfr x F- mating -> only a few recombinants… How to select them??
Must eliminate Hfr (donor) & F- (nonrecomb. recipients)
Method
Hfr strain has alleles not present in F- strain: selected markers
Ex: leu+
F- strain has an allele not present in Hfr: counter-selected marker
Ex: strR= streptomycin resistant (str5 = susceptible)
Genes to map: Hfr=leu+ … str5 x F- + leu- … strR
Resulting bacteria:
Donors = Hfr leu + …str5
Nonrecombiant recipients = F- leu- … strR
Recombinants = F- leu+ … strR
Only recombiants will grow: F- leu+ … strR

Hfr can convert back to F+ … but in the process, plasmid may pick up genes from donor genome.

Partial diplioid bacterium = merozygote
==Transformation:== small pieces of extracellular (exogenous) DNA are taken up by bacterial cell and (often) integrated stably into the chromosome. Another method of recombination. Bacteria must be competent - in bacteria, transient state during which the cell can bind and internalize exogenous DNA, making transformation possible. Outcomes: entry of foreign DNA into recipient cell and/or recombination between foreign DNA and its homologous region in recipient chromosome.
==Electroporation:== an artificial version of transformation using electrical current ot temporarily increase permeability of the bacterial cell membrane. Extensively used in cloning to introduce vectors into E. coli host.

Process of transformation

==Heterooduplex==: region that contains 1 host strand and 1 mutant strand, from different sources. Usually contains some mismatch of base sequence -> activates DNA repair
DNA incorporated in transformation: up to 10-20 kb (encompassing several genes)
Cotransformation: nearby genes may be recombined in a single transformation event
Close genes (maybe) require only a single event
Distant genes require multiple independent events
DNA from a bacterium with genotype a+ b+ c+ is used to transform a bacterium that is a- b- c-. The numbers of each transformed genotype are below. What can you say about the relative positions of the genes? (i.e., which genes are closer together, which are farther apart?)

Bacteriophages (phages) are viruses that infect bacteria (host)

Bacteriophages life cycle

Lytic cycle- because ultimately the host cell is lysed
Lysogeny: phage DNA integrates into bacterial genome (= lysogenic cycle) virus=prophage, bacterium = lysogenic
Viral DNA passes through the generations. Replicated along with host DNA. no new viruses produced.
Some environmental cue starts the lytic cell.
Transduction: phage-mediated bacterial DNA transfer/recombination. During step 4 of lytic cycle, a phage accidentally incorporates some bacteria DNA into its head (“defective phage”). When that phage infects another cell, the bacterial DNA gets transferred and integrated into recipient chromosome.

Generalized transduction: bacterial recombination is mediated by bacteriophage
Cotransduction: two genes are close enough to be transduced simultaneously. Two independent transduction events may occur if genes are not close enough.
Mapping via transduction
Closely linked genes may be cotransduced (cotransduction). Mapping via transduction is just like transformation mapping, so no further details needed here.
Mapping in bacteria:
Three different methods
All rely on recombination
Concept of selective plating

Chapter 13: Developmental Genetics
Development: attainment of a differentiated state by all cells organism (except stem cells)
Differentiation: acquisition of specialized cellular characteristics. Different cells contain different proteins because they express different genes.
Cells express different genes because they contain different combinations of transcription factors.
Cells end up with different transcription factors because transcription factors are proteins, thus all the products of genes. Cells have different transcription factors through differential expression of transcription factors genes (sort of a chicken & egg problem).
Cells become restricted in fate through a series of decisions, usually binary.
Fate: the capacity to differentiate into a particular cell type.
Simple fate decisions:
Sex determination (binary: two choices)
Separation of the germline and soma (also binary)
Plants: leaves or flowers
Blood stem cells develop into ~20 different cell types
Fate decisions are “switches”: patterns of gene expression are usually selected by either/or mechanism.
Variable gene activity hypothesis: differentiation is accomplished by activating and inactivating genes at different times in different cell types. Assumption: each cell contains the entire genome.
Each differentiated cell type in the adult has a distinct pattern of gene expression.
Fertilization: egg and sperm fuse to form a single totipotent cell – the zygote. Totipotent cells can differentiate into any specialized cell type in embryo or outside embryo (e.g., placenta)
Cell division continues -> blastula -> then totipotent cells begin to specialize.
From blastula, 30-40 embryonic stem cells (ESCs) form – pluripotent. Pluripotent cells can differentiate into any specialized cell type in embryos only. (i.e., NOT the placenta)
Overview of Drosophila development:
Cytoplasm of fertilized egg is organized into a series of protein gradients produced by
expression of maternal genes.
These gradients play a key role in determining developmental fates of zygote cell nuclei.
Fertilization -> (B) a cell with multiple nuclei (syncytium)
© Zygote nuclei divide and migrate to the periphery of the embryo. (D) cellularization
~6000 cells at cellularization (~3 hrs). Cells are now committed to a fate (though none have
differentiated). 20 hrs: larva hatches, 14 segments. Body plan was determined in the first 3 hrs.

Cell fate is specified independently along the anterior-posterior axis (head-to-tail) and dorsal-ventral
axis .
Cell fate is specified gradually, from coarse to fine.
Multiple, progressive changes in gene expression.
Genes that affect Drosophila development
Anterior-posterior axis
Segment number
Maternal effect genes (bicoid, nanos)
Gap genes (zygotic)
Pair-rule genes (zygotic)
Segment polarity genes (zygotic)
Segment identity
Homeotic selector genes (zygotic)
Dorsal-ventral axis
Many genes (maternal and zygotic), not discussed
Genetic control of Drosophila development
Two levels of control: maternal-effect genes and zygotic genes
Maternal-effect genes
TFs and mRNA; proteins made after fertilization
Products distributed in a gradient or concentrated in specific regions of cell -> polarity
Regulate gene expression - activate or repress expression of zygotic genes
Zygotic genes
Transcribed in nuclei of embryo
Transcribed in specific regions in response to distribution of maternal-effect proteins
Deleterious recessive mutations in homozygotes lead to embryo lethality.
Maternal-effect genes
Proteins and mRNA determine anterior-posterior (A-P) axis
Patterning of anterior parts (head, thorax): bicoid (bcd)
Patterning of posterior abdominal segments: nanos (nos)
In Drosophila, mutants denoted using gene name. So for bicoid:
Wt = +
Mutant = bcd
Bicoid mutations in mother: embryos lack head and thoracic segments.
Maternal effect: genotype of mother determines embryonic phenotype. Examples:
Female bcd/bcd x male +/+
All embryos die with anterior defects
Female +/+ x male bcd/bcd
All embryos are normal
Explanation for maternal effect phenotype: gene is transcribed during oogenesis, but protein is
Necessary for embryogenesis.
Bicoid gene transcribed during oogenesis, and bcd mRNA is localized in the anterior egg.
Bicoid protein translates after fertilization and forms a protein gradient.
Bicoid protein is a transcription factor that activates anterior gap genes.

Cellularization: when embryo cellularizes, each cell receives any proteins present in the cytoplasm in
Position. In this way, the concentration gradients of maternal-effect proteins present in early egg will
Become differences in concentration of each protein within a cell.
Genetic control of Drosophila development
Zygotic genes show “normal” mendelian genetic behavior
(embryonic genotype = embryonic phenotype).
+/kni x +/kni
¼ +/+ normal phenotype
½ +/kni normal phenotype
¼ kni/kni lethal (embryos die with abdominal defects)

Segmentation genes: divide embryo into segments (mutants lethal). Identified and classified based
on mutant phenotype:
Gap genes: mutations delete a group of adjacent segments and cause large gaps in normal
body plan. Establish broad multi-segment regions of embryo that ultimately become head,
Thorax, and abdomen.
Regulated by maternal effect gene products (e.g., bicoid)
Produce TF to regulate next set of genes (pair-rule)
Hunchback (hb), Kruppel (Kr), knirps (kni)
Embryos homozygous for gap gene mutations lack contiguous blocks of segments.
Gradients of maternal proteins establish domains of gap gene expression.
Ex: bicoid is a transcription factor and activates gap genes in a concentration-
Dependent manner.
High bicoid concentration = expression of hunchback (hb)
Below a minimum concentration of bicoid, hb is no longer expressed, but
Knirps (kni) is.
Pair-rule genes: mutations affect every other segment and eliminate a specific part of the
segment. Divide embryo into 7 stripes about 2 segments wide.
Stripes are precursors to segmental body plan
Turned on by gap genes; control expression of next set of genes (segment polarity)
At least 8 pair-rule genes act to divide embryo into a series of overlapping stripes
Embryos homozygous for pair-rule genes lack alternating segments
Segment polarity genes: mutations cause defects in a portion of a segment (e.g., wingless).
Regulate anterior-posterior spatial pattern of differentiation within each segment of embryo.
Regulated by transcription factors encoded by PR genes
Divide embryo into 14 segments
Gene products control cellular identity within each segment and establish AP polarity
Within each segment.
Segment boundaries are further sharpened by inhibitory interactions between SP
Proteins.
Each AP position now defined at 1-cell resolution by expression of unique set of gap,
PR, and SP genes.

Segmentation genes in vertebrates
Vertebrate homologs to many Drosophila segmentation genes
Runt mutations (pair-rule gene in Drosophila)
Runt mutations in humans: cleidocranial dysplasia
Summary: regulation of development in Drosophila

Maternal effect genes determine the anterior-posterior axis and induce gap genes
Gap genes define several broad areas and regulate…
Pair rule genes, which refine segment locations and regulate…
Segment polarity genes, which determine the boundaries and anterior-posterior orientation of
Each segment.
Together, the gap, pair rule, and segment polarity genes control expression of the Hox genes,
Which define the identity of each segment.

Homeotic (selector) genes control what structures are formed in a body segment.
Segment identity
Activated as targets of zygotic genes
Determine which adult structures will be formed by each body segment – antennae, mouth parts,
Legs, wings, thorax, abdomen.

Homeotic mutants: one segment forms a structure of another segment
Hox genes in Drosophila
Two clusters on chromosome 3:
Antennapedia (ANT-C) complex: 5 genes specify structures in head and first 2
segments of Thorax.
Bithorax (BX-C) complex: 3 genes specify structures in posterior portion of second
Thoracic Segment, entire third thoracic segment, and abdominal segments.
Hox genes are a subset of homeobox genes: genes that contain a homeobox and regulate large-scale
Anatomical features in early stages of embryonic development.
@@Homeobox@@: DNA seq domain ~180 bp, encodes a homeodomain protein product (~60 AAs) –
TF that binds to DNA to regulate gene expression
“Hox” technically does not equal “homeobox”
Expression of Hox genes is collinear with the A-P organization in embryo

Many Hox genes act by producing protein products (transcription factors) that repress or activate other developmental genes.
Ubx represses genes involved in wing formation.
3rd thoracic segment: normally 1 pair of legs, 1 pair of highly reduced wings for balancing.
abd-A represses limb formation by repressing the gene distal-less (DII)
A number of genes that control expression of Hox genes in Drosophila have been identified. One of these homozygous mutants is extra sex
Combs. This gene is normally expressed throughout the embryo. In esc- mutants, some of the head and all of the thorax and abdominal
Segments resemble the last abdominal segment.
@@Synpolydactyly@@: rare limb deformity characterized by co-presentation of syndactyly (fusion of digits) and polydactyly (more than typical
Number of digits) caused by mutation in HOXD13

Which function earlier in development: %%maternal-effect genes%% or zygotic genes?
Which class of genes controls the development identity of segments along the anterior-posterior axis?
Maternal-effect genes
%%Homeotic genes%%
Segmentation genes

Plants evolved developmental regulatory systems that parallel those of animals.
Development patterns evolved independently in animals and plants.
Flowers develop from a group of undifferentiated cells (floral meristem)
Various floral homeotic genes control development of the 4 organs
Some of these genes are MADS-box genes – no sequence homology with Hox genes!
==Binary switch genes==:
Two alternative developmental fates for cell
Initiate complete development or organ or tissue type
Binary switch genes in Drosophila:
Wt allele of binary switch gene programs eye formation instead of antenna
Mutant allele eyeless: eyes are reduced in size
Another kind of development gene resembling a homeotic gene in that it is a transcription factor with hundreds of target genes
1990s: discovery of eyeless gene in flies and Pax6 in mice, same gene, different name
Encodes a transcription factor necessary for lens development.
Walter Gehring inserted mouse Pax6 into the genome of a fly in the leg.
Pax 6 gene was turned on by regulatory factors in fly leg.
Compound eye (III) formed on leg of fly
In cavefish, pax6 gene expression is reduced in cave dwellers. Eyes start to develop, but then they degenerate.