Flashcards in DNA genetics Deck (57):
How goes the information on DNA determine protein structure?
dsDNA has a template strand (3'-5'), and an RNA-like strand (5'-3'). The template strand has a complimentary sequence to the RNA like strand of the mRNA-the sense strand or + strand. RNA polymerase goes from 3' to 5' on the template strand producing mRNA going from 5' to 3' identical to the RNA-like strand. The 5' start codon of mRNA when translated becomes the N-terminal end of the protein transcript, 3' end the C terminal.
What are long non-coding RNA (IncRNA)?
Long non-coding RNA (>200 bp) has been found to modify gene expression that can be inhibited by small interfering RNA. Protein coding RNA accounts for only 1/5 of transcription.
IncRNA occurs in osteoarthritis and encourages metalloproteinase production and inhibition results in increased aggregation.
Why are some loss of function mutations
A dominant allele usually codes for a functional protein, and other alleles are recessive as the altered protein is less functional. Loss of function occurs in a diploid organism if the gene product of the wild type allele is insufficient for normal function, resulting in an abnormal phenotype due to the non functional recessive allele. The mutation is due to haploinsufficiency eg hypercholesterolemia.
If the mutation antagonizes the normal product the genotype is called a dominant negative mutation eg Huntington's chorea.
How does miRNA interfere with DNA transcription?
The miRNA-inducing silencing complex (RISC) is generated by Drosha binding of primary transcript and cropping, exporting to cytoplasm where Dicer crops dsRNA then degrades one strand producing miRISC, which pairs with messenger RNA causing cleavage and mRNA destruction or translation inhibition. if only partly complementary.
What role might endogenous retroviruses play in gene expression?
Endogenous retroviruses present in eukaryotic genomes may function as promoters and rarely expressing their own genes. Salivary amylase owes it's expression in salivary tissue to an endogenous retroviral promoter. Placental function depends upon similar retroviral effects.
ERV account for about 9% of mammalian DNA.
How many types of transposable elements are there in the human genome ? How common are they?
There are 2 categories transposable elements:-
a) SINEs (short interspersed nuclear elements) 1-5Kb (U3-R-U5 structure at both ends of retroviral sequences), have poly A tail, 2 million per genome, ~11% human genome.
b) LINEs (long interspersed nuclear elements), contain ORF1, and pol genes, poly A tail, 6-8 kb long, 1 million per genome. 21%.
c) HERVs (Human Endogenous Retrovirus), gag, pol and env genes plus LTR's at both ends. 1-11 kb long, 600,000/genome, 8%.
2) DNA transposons, have no RNA phase, contain 1 transposase gene flanked by inverted repeats, 2-3 kB long, 400,000/genome, 3%.
Some LINES are autonomous (100/genome), can move by themselves. All SINES are non autonomous and require expression of LINE genes. Almost all LINE and SINE insertion points are the same in all humans with 8000 mobilized during human history.
Alu elements are primate retrotransposons, 300 bp, (SINE), make up 11% human genome, of which 7k are unique to humans. W, alu element.
What is the difference between ordinary PCR and ms-pcr?
Ordinary PCR produces amplicons - more than one copy of a genetic fragment of a target sequence.
ms-pcr refers to methylation-specific polymerase chain reaction which detects methylation sites. Treatment with bisulphite changes non methylated cytosine to uracil, methylated cytosine is resistant.
What is a CpG motif?
CpG motifs refers to a DNA sequence where cytosine is 5 prime to quanine. The cytosine can be methylated to methylcytosine in mammals by DNA methyltransferases which turns the gene off.
What is the difference between a promoter and enhancer?
A promoter is a region of DNA that initiates transcription of a particular gene. They are located near the start site on the same strand and upstream of the 5' region of the sense strand and can be 100 to 1000 base pairs long. TATA box binding proteins (TBP) and TBP associated factors (TAF's) bind the promotor sequence then attract pol II to start transcription.
An enhancer is a DNA sequence that is trans activated by transcription factors, on the cis-acting enhancer sequence, located up to 1 million base pairs (1Mbp) away upstream or downstream from the start site. TF binding attracts mediator proteins that complex with the pol II complex by looping the DNA strand. Transcription increases by changing the polymerase and attracting co-activators which remove nucleosomes.
Insulator sequences work by fixing loop formation that prevents enhancer looping.
How does the REST/NSRF repressor protein work?
REST/NSRF (RE1-silencing transcription factor/neuron-restrictive silencer factor) is a repressor protein, 1,097 aa with 9 zinc finger motifs, that associates with mSin3 (interaction domain for TF suppression) and CoREST (nerve differentiating factor) to silence many transcription factors by deacetylating and demethylating specific sites on histones. Active in neurons, and inactive in neoplasia-small cell lung CA.
Where does the internal ribosomal entry site (IRES) usually insert?
An internal ribosome entry site (IRES) is a nucleotide sequence that permits ribosomes to begin at that point on mRNA. IRES are often located on the 5' UTR of RNA viruses, and in mRNA of animal genes involved in mitosis, and apoptosis to ensure transcription.
What is the function of terminal deoxynucleotidyl transferase? T
Terminal deoxynucleotidyl transferase adds N-nucleotides• to the V, D, and J exons during antibody gene recombination thus enabling junctional diversity. DNTT gene. The gene is present in immature T and B cells and multipotent hematopoietic stem cells.
How does mIRN-21 micro RNA lead to tumor suppression?
mIRN-21 is a cytoplasmic micro RNA (72 bp) originally transcribed from the an intron of the vacuole membrane protein gene. The product is a stem-loop precursor mRNA which is cut by the endonuclease Drosha, exported to the cytosol, cut again by Dicer and its other strand is then degraded. The final micro RNA targets tumor suppressor genes (3' UTR) such as PTEN, Bcl2, IL-12p35, and many others.
What part of the endogenous retrovirus genome is capable of altering host gene expression?
Long terminal repeats are at both ends of endogenous retroviruses and serve as viral integration sites into new areas of the genome. They frequently contain promoters, and enhancers capable of influencing transcription of nearby host genes.
How does Sirtuin 1 act on chromatin?
Sirtuin 1 is an NAD-dependent deacetylase related to the silent mating type information regulation 2 homolog 1 of S. cerevisiae. It reverses epigenetic DNA changes favoring gene expression-histone acetylation thereby reducing gene transcription. It may also silence LINES thereby reducing gene hopping induced mutations in the elderly.
Low levels in progeria
What is the difference between prokaryotic and eukaryotic gene transcription?
Prokaryotic transcription uses a 5 unit polymerase, transcription sequence are located near the promotor, starts with a ribosomal binding site, formyl methionine loads the ribosome, can begin translation during RNA transcription, can transcribe at multiple sites along the mRNA.
Eukaryotic transcription requires nucleosome unwinding, promotor attachment, usually has an untranslated initial sequence, a methionine start site which extend the 5' UTR, requires different polymerases for different types of genes, needs mRNA splicing to remove introns from primary transcript, attaches a 5' methylated cap and a 3' poly A tail, cannot begin translation until transcription and splicing are complete, only one ribosome may attach at a time, the stop codon extends to 3' UTR.
What controls the progression from one cell stage G1 to S?
Satisfaction of checkpoints and progression of cyclin-dependent kinase (CDK 1-4) attached to various cyclins (a-E) which phosphorylates Rb protein (protein product of retinoblastoma) component of the Rb-E2F complex. Phosphorylation frees E2F transcription factors to express the genes necessary for DNA synthesis (DNA polymerase, thymidine kinase, and dihydrofolate reductase). Phosphorylation is inhibited if DNA has been damaged.
How did Mendel develop the plant material on which to propose his laws of heredity?
He started by breeding plants that consistently had progeny in which all members showed the same trait (homozygous). By crossing these plants with others of different traits the F1 generation showed mixed results in a consistent pattern. He noted which traits were dominant or recessive and independent assortment when studying multiple traits. Using homozygous parents who developed heterozygotes F1 generation necessarily had fixed proportion of different traits.
How are dominant and recessive traits expressed in family trees?
Dominant traits always include one affected parent, showing a vertical pattern of inheritance. Two affected parents, and heterozygotes can occasionally have a normal child.
Recessive traits are expressed in all children of affected parents. Horizontal pattern develops with affected children belonging to the same generation. Vertical inheritance pattern occurs if the recessive trait is common in that population. Affected children need not have affected parents but are more common in consanguineous unions.
How have Mendel's laws been adjusted?
Dominance needed adjustment for degree of dominance.
Incomplete-1:2:1 red:pink: white
Codominance produces phenotypes 1:2:1. peas-
2->multiple alleles produce phenotypes in a series of 3:1
gain-of-function alleles that are hypermorphic (achondroplasia-over expression of FGFR3 that activates TK and stops bone growth ) , neomorphic (huntington), or antimorphic (antenna to leg).
Some recessive alleles are lethal ->2:1 ratios.
Some genes influence several traits-pleiotrophy.
Genes vary in expressively (degree to which a specific genotype is expressed in a phenotype), and penetrance (degree to which those with the same genotype show the same phenotype).
How may 2 genes interact in a dihybrid F1 cross in the F2 generation?
4 distinct phenotypes-9:3:3:1 seed coar color peas-brown, tan, gray, green. 4x4 table AABB->aabb with 16 cells
Complementary: one dominant allele of each gene necessary-9:7 sweet pea flower color purple:white.
Recessive epistasis: homozygous recessive blocks both alleles of 2nd gene 9:3:4 labs black brown yellow
dominant epistasis type 2: dominant allele B (prevent pigment deposition, b-wild type deposits color) hides both alleles of A gene 12:3:1 squash color-white:yellow:green. green=bb if no A (white)
redundancy: only 1 dominant allele of either gene produces phenotype 15:1 Maize leaf
How are open reading frames discovered within genomic DNA?
Isolated clones of genomic dsDNA are searched in both directions 2x3 transcripts for stop codons. Any sequence in any direction that codes for 7 aa in a row without a stop codon is an open reading frame.
What limits the use of cDNA as a way to find genes?
cDNA needs to come from tissues that express the gene of interest which depends on the tissue studied and its metabolic activity.
What genomic DNA sequences show the greatest homology to humans among various other species.
Protein coding sequences are highest, and introns much less so. Intergenic sequences are not conserved.
what is the relationship to Lamin A and progeria?
LMNA gene, expresses prelamin protein which is farnesylated (CH3-C=C(CH3)2) and attaches to the nuclear membrane. Prelamin is defarnesylated and lamin released which stimulates sirtuin. When prelamin is not released progerin-farnesylated prelamin interferes with nuclear membrane resulting in progeria. Sirtuin stimulation by lamin A decreases, which contributes to progeria.
How long-ago (mya) did humans share a common ancestor with other vertebrates-Zebrafish, chickens, dogs, mouse, monkey?
Our last common ancestor with zebra fish occurred 400 million years ago, with chickens 300, with cow, horse, dog 90, with mouse 75, and with monkeys 25.
What genetic abnormalities account for thalassemia?
Alpha and beta globulin genetic abnormalities either in the locus control region (LCR) or within individual globulin genes resulting in complete or partial decrease in normal function. Alpha genes (Ch 16) may have 0 to 4 genes ( (4-100%, 3-75%
heterozygous 50%, 1-25% severe anemia, 0-lethal).
Beta genes (Ch 11) may be replaced by embryonic or fetal hemoglobin which are on same chromosome, or none if locus of control is affected-severe anemia.
What is the relationship between type of polymorphism, size, and frequency per kilobyte?
Single nucleotide polymorphisms (SNIP) are 1 bp in size and occur 1/kb.
Insertion/deletion (DIP or indel) are 1-100 bp, every 10 kb.
Microsatellites or simple sequence repeats (SSR) are 1-10 bp repeat unit, every 30 kb. Used in analysis for kinship, population, gene duplication or deletion, marker assisted selection, and fingerprinting.
Copy number variant (CNV), variable number of genes copied and inserted together, are 10 bp - 1 Mb in size every 3 Mb.
What special problems do copy number variants (CNV) produce during cell division?
Malalignment and unequal crossing over result in recombinant products-new alleles that have more or fewer repeating units than the parental type. CAG repeats in Huntington disease 30 normal->50 in late onset, 100 in early onset.
In the polymerase chain reaction (PCR) where do the priming oligonucleotides bind and in what direction does the polymerase move?
dsDNA containing the gene of interest is heated, both strands are present. Two primers (16-30 bp oligonucleotides) bind at the 5' end of opposing separated DNA strands and DNA polymerase moves in the 5' to the 3' direction, replicating the gene of interest in different directions. After 3 rounds of replication, most strands have primer beginnings and replicate only the DNA between primers in subsequent replications. After 30 repetitions over 1 billion copies are made.
How good is DNA fingerprinting?
DNA fingerprinting identifies 13 known SSR (Simple sequence repeats) loci such that the chance of two people (except twins) having the same pattern is less than one chance in 10 trillion.
What are the relative merits of positional cloning, pedigree analysis with LOD scores, and whole genome sequencing when attempting to determine a genetic mutant?
Positional cloning using snips and developing LOD scores defines the area of interest but whole genome sequencing permits direct analysis of gene structure provided both parents are also studied.
The search area can be reduced by examining only the exome, selecting only missense mutations, selecting only variants not previously reported in databases, selecting variants that are X-linked or display recessive pattern, selecting variants that change evolutionarily conserved amino acids within proteins, selecting for variants in genes that are not frequently mutated in the general population, and then selecting genes known to be mutated in other genetic diseases of some relevance.
What is the difference between hetero and euchromatin?
Heterochromatin is condensed (nucleosomes 100 A wide -> fiber 300A) and transcriptionally inactive as opposed to euchromatin which is open and more accessible to promoters. Heterochromatin can increase along a chromosome causing position-effect variegation (PEV). Effect is limited due to barrier insulators.
One X-chromosome in females becomes a bar body to the presence of Xist gene that transcribes a long non-coding RNA which induces condensation of the chromosome. The Y-chromosome contains large sretches of heterochromatin.
What are the essential features of an artificial chromosome
Artificial chromosomes are constructed from at least 100 K bp, that contain a centromere, yeast origin of replication center, and 2 telomeres.\
Why do telomeres shorten?
Telomeres tend to shorten because DNA polymerase needs a primer at the 3' end of each chromosome, and after replication the DNA is short by the length of the primer (?6bp). Stem cell chromosomes activate telomerase which can make up the difference, adding DNA based on the presence of RNA in the telomerase-6 bp. Telomeres in nondividing cells are protected by loop construction (Shelterin complex) which prevents enzymatic destruction.
How do centromeres function to permit variable chromatid separation?
The centromere in eukaryotes accommodates multiple microtubules which are attracted to proteins that recognize special sequence repeats of the centromere. Motor units on the microtubules apply tension to the chromosomes which separate after cohesin undergoes proteolysis once the checkpoint for proper separation of all chromosomes has been completed. Prophase of meiosis 1 allows crossing over between homologous chromosomes but centromere does not split (cohesin intact). Centromeres split during anaphase of mitosis 2 prior to gamete formation where crossing over results in variable chromatids, unlike either parental form.
Do bacteria contain insertion sequences?
Insertion sequences ( IS) and bacteria are 700 to 5000 base pairs includes a transponase gene with inverted repeats at both ends. a composite transposable element (Tn)also carries a gene. They perform like d transposable elements in eukaryotes.
How is horizontal transmission of genetic material accomplished in bacteria?
Transformation occurs when the DNA release by dying bacteria are taken up by a live one.
Conjugation inserts new DNA from one bacteria to another by a connecting tube.
Transduction occurs when a bacteriophage inserts genetic material directly into a bacterium.
Can plasmids integrate into bacterial DNA?
Yes, a bacterium with an integrated plasmid called an Hfr bacterium (high frequency of recombination), and the extrachromosomal plasmid capable of integrating is called an episome.
Episomes are sites where new genes can be inserted.
How do mitochondria vary between species?
Mitochondria varied by length of genome, presence or absence introns and circular versus straight. mRNA is edited via RNA editing (editosome), and DNA code somewhat different.
Why are mitochondrial DNA defects expressed variably?
The egg may divide mitochondria unevenly, some with a mutant variety and some mostly normal. A homoplastic genotype contains mostly one variety or another, and a heteroplasmic genotype contains both. The homoplastic variety is usually grossly abnormal and the heteroplasmic severity depends on the dose of mutant genotype.
How do repressor factors work?
Repressors bind the enhancer sequence and attract corepressors which may block binding to the promotor or increase nucleosome formation, hiding the promoter.
What is a CpG Island and what do they do?
CpG islands are sequences rich in CpG dinucleotidea of 100 to 2000 Kbp that are usually not methylated. Methylation results in increased nucleosome formation which can repress nearby promoters, via methyl-CpG binding proteins (MeCP's). pattern of methylation is preserved following mitosis accounting for imprinting.
How is the Prader-Willie syndrome related to Angelman syndrome?
The Prader-Willie syndrome (small hands and feet, underdeveloped gonads, short stature, mental retardation, compulsive overeating) is associated with a small chromosome 15 deletion, inherited from father. The same lesion inherited via the mother results in Angelman syndrome (red cheeks, large jaw and mouth, prominent tongue, mental and motor retardation). At least two genes in the depleted area are differentially imprinted. PrinteriWilliie syndrome has the mothers wild type gene imprinted, and vice versa for the Angelman syndrome.
What are the main types of small RNAs in eukaryotes?
All are 21-30 bp, but vary in sequence structure.
miRNAs block translation and destabilize mRNA.
siRNAs recruit histone-modifying enzymes resulting in heterochromatin formation.
piRNAs ( origin pIWI interacting RNA, from PIWI- P element induced wimpy testes in drosophila, protein maintains embryonic potential)) are RNAs that bind piwi protein which complexes to DNA transposable element that degrades transposable elements or facilitates histone modification that inhibits transposable element transcription.
What diseases are caused by triplet repeats?
PolyQ diseases code for glutamine, Affect the protein product, and cause Huntington disease. Non-PolyQ diseases include fragile X syndrome where > 55 CGG repeats result in disease.
What are the different types of gene rearrangement?
Rearrangement may result in deletion, duplication, or inversion of genes on the same chromosome, or translocation of gene sequences onto other chromosomes.
All are responsible genes identified in a mutant?
Transgene tagging (fluorescent protein), positional cloning (mapping), genome sequencing may identify likely candidates, and rescuing the mutant or inducing the mutant will verify the responsible gene.
Pathway identification and ordering genes, eventually explains mutant development.
What are the major types of genes involved in early embryogenesis?
Maternal effect genes (bicoid-anterior mophogen) and nanos (posterior morphogen) organize the fertilized egg, zygotic genes determine segment number and polarity: Gap genes and segment polarity genes. Pair rule genes that determine segment (14) polarity (wingless, hedgehog). Homeotic genes then specify segment identity.
What is the unique characteristic of homeotic genes?
Homeotic genes (Hox) are arranged in the order they are expressed throughout the animal kingdom. Each one produces a DNA binding protein-helix turn helix transcription factor.
How is the frequency of the phenotype in a population related to the frequency of alleles determining that phenotype?
The Hardy-Weinberg relationship of p2 plus 2pq plus q2 =1 holds if two alleles are involved provided the population involved is infinite, genotype does not determine choice of mate, no new mutations develop, no migration into or out of the population, and genotype does not determine fitness.
Harding and Weinberg proposed their law in 1908.
What is the difference between autosomal and X-linked genes affecting the phenotype?
Autosomal genes appear in the next generation in proportion to their frequency whereas it takes 4 generations for a new X-linked gene to appear in the expected frequency as male and females contain the gene in different proportions.
What accounts for differences in genotype frequency in different human populations?
Genetic drift develops due to small founder populations (population bottlenecks) where by chance some genes dropout and others become fixed. it may take five generations for a new adventitious mutation to become fixed in a small population (20) or 200 generations in a large population.
How many thousand years ago did migration out of Africa result in different populations?
60,000 years ago Australia was settled, 40,000 years ago some of the Australian migration moved into northern China, and new migration from Africa moved into the Middle East. 30,000 years ago some of the Middle East people moved into India, Burma, northern Europe and by 20.000 years were in South America. 10,000 years ago some from the middle east moved into southern Europe some from India moved into southern China and Indonesia, some from northern China moved into North America ( second migration).
How does one distinguish between nature and nurture?
By comparing the variability of a phenotype within a genotype in terms of variance the amount of variance can be divided between that due to nature, and that due to nurture. Broad sense heritability H2, is the ratio of variance in the population as a whole to that in a specific genotype. Narrow sense heritability h2 measures the total variance due specifically to the variance of an added genetic component. Heritability therefore refers to population not individuals. Unexplained variability is attributed to environment.
How are genes floxed and for what reason?
lox P sites (locus of X-over P1 from bacteriophage P1, 34bp, asymmetric 8 bp sequence) are inserted on each side of the gene of interest. A similar construct with different P site and a replacement gene (with P site flanking) are also inserted. Viral recombination protein inserted elsewhere or in parent (cre Causes Recombination, 343aa integrase,4 subunits, 2 domains) is then activated causing the new gene of interest to replace the old one.