Bio 218 Exam 3 (8.3 - 12)

Two main themes underlying the observations on chromosomal changes

1. Karyotypes generally remain constant within a species -most genetic imbalances result in a selective disadvantage 2. Related species usually have different karyotypes -closely-related species differ by only a few rearrangements -distantly-related species differ by many rearrangments -correlation between karyotypic rearrangments and speciation

Two types of events that reshape genomes

1. rearrangment - which reorganize the DNA sequences within one or more chromosomes 2. changes in chromosome numer involving losses or gains of entire chromosomes or sets of chromosomes

Nucleosome supercoiling model of higher-order packaging

100 A nucleosomal chromatin is compacted into 300 A fiber by supercoiling H1 plays special role in formation of the superhelix, without causes 300 A to unwind to 100 A

The nucleosome core is an octamer of two each of histones H2A, H2B, H3 and H4

160 bp of DNA wraps twice around a nucleosome core 40 bp of linker DNA connects adjacent nucleosomes Histone H1 associates with linker DNA as it enters and leaves the nucleosome core

Action of cohesin during meiosis II

After entry into metaphase II, shugoshin is removed and centromeric cohesin is degraded

Constructing yeast artificial chromosomes (YACs)

Artificial chromosomes require three key elements: centromeres, telomeres, and origins of replication Consequences of deleting key elements: -no centromere, no telomere - replication but have segregation errors -no telomere - replicate, degrade if linear

Action of cohesin during meiosis I

At anaphase I, cohesin along chromosome arms in enzymatically cleaved but cohesin at centromeres is not cleaved Shugoshin protects centromeric cohesin from degradation

Nonsense suppression

Cells with nonsense-suppressing mutation in a tRNA gene can survive if two conditions coexist: 1) cell must have other tRNAs that recognize the same codon as the suppressing tRNA recognized before mutation altered its anticodon (without has no way to inser proper amino acid in response to that codon) 2) the suppressing tRNA must have only a weak affinity for the stop codons normally found at the ends of mRNA coding regions (if not, the suppressing tRNA would wreak havoc in the cell, producing an array of aberrant polypeptides that are longer than normal) Cells guard against this by having termination depend on two stop codons in a row A second, nonsense suppressing mutation in the anticodon of a tRNA gene allows production of a (mutant) full-length polypeptide

Nonsense mutations

Change codon that encodes an amino acid to a stop codon (UGA, UAG, UAA) change an amino acid specifying codon to a premature stop codon result in the production of truncated proteins lacking all the amino acids between the amino acid encoded by the mutant codon and the C terminus of the normal polypeptide. mutant polypeptide will be unable to function if it requires the missing amino acid for its activity earlier it occurs, more devastating

Chromosomal DNA and proteins

Chromatin is the generic term for any complex of DNA and protein found in a nucleus of a cell Chromosomes are the separate pieces of chromatin that behave as a unit during cell division Chromatin is ~1/3 DNA, ~1/3 histones, ~1/3 non histone proteins DNA interaction with histones and nonhistone proteins produces sufficient level of compaction to fit into a cell nucleus

Ch. 12

Chromosomal Rearrangements and Changes in Chromosome Number

Kinetochores assemble at centromeres

Chromosomes attach to microtubules of the mitotic spindle at the kinetochore Chromatin is packaged differently at the centromere -H3 is replaced by the histone variant CENP-A -CENP-A nucleosomes act as scaffolds for many kinetochore proteins

Action of cohesin during mitosis

Cohesin is a protein complex that holds sister chromatids during metaphase; encircles the two double helixes of the sister chromatids to keep them together At anaphase, cohesin is enzymatically (separase) cleaved and sister chromatids are released from each other rings are scattered along the length of the chromosome but mainly at the centromere

The structure and function of the eukaryotic chromosome

Copy cate is a clone of rainbow: all cells have identical DNA, each cat has many types of cells, the cats are dissimilar in many phenotypes Chromosomes support the packaging, replication, segregation, and expression of genetic information

Aberrant crossing-over can cause all four types of chromosomal rearrangment

Crossing over between repeated sequences on homologous or nonhomologous chromosomes the repeated sequences may be tandem repeats like simple sequence repeats (SSRs) or transposable elements (DNA sequences whose copies move from place to place, so that a single genome may accumulate hundreds of thousands of copies of such an element crossovers between repeats of same sequence at two locations on same chromosome result in deletion if in same orientation, or an inversion if in opposite orientation. if two homologous chromosomes misalign at repeated sequences and cross over the result may be a deletion and a duplication crossovers at repeated sequence on two nonhomologous chromosomes generate reciprocal translocations

X-ray crystallography of a nucleosome

DNA bends sharply at several places as it wraps around the core histone octamer Base sequence dictates preferred nucleosome positions along the DNA

Nucleosomes are disassembled and reformed during DNA replication

DNA is packaged in nucleosomes within minutes of synthesis A tetramer of H3 and H4 associates with DNA, followed by two dimers of H2A and H2B New nucleosomes are composed of recycled and new histones Chromatin is open to histone modification just after replication

Deletion loops form in the chromosomes of deletion heterozygotes

Deletion loop: an unpaired bulge of the normal chromosome that corresponds to the area deleted from the other homolog Recombination between homologs can occur only at regions of similarity No recombination can occur within a deletion loop Consequently, genetic map distances in deletion heterozygotes will not be accurate, because fewer crossovers can occur

Four types of chromosomal rearrangment

Deletion: removing base pairs Duplication (insertion, adding bp) Inversion (180 rotation) Translocation - two nonhomlogous chromosomes exchange parts

Fluorescent in situ hybridization (FISH) is used to characterize genomes

Depends on hybridization between metaphase chromosomes and a labeled DNA sequence -chromosomes are spread on a glass slide and denatured to make them single stranded -a DNA sequence is labeled with a fluorescent tag to make a probe -the probe hybridizes to chromosomes at complementary regions FISH: provides a convenient bridge between the low resolution of a karyotype and the ultra-high resoltuion of a complete genomic sequence, allows to find locations of specific DNA sequences with respect to the chromosomes in a karyotype

DNA breakage can cause all four types of chromosomal rearrangement

Double strand breaks followed by nonhomologous end-joining Can be due to x-rays

Segregation of condensed chromosomes depends on cetromeres

During anaphase of mitosis and meiosis II, sister chromatids must segregate to different daughter cells During anaphase of meiosis I, sister chromatids do not separate and homologous chromosomes segregate to different daughter cells Centromeres have two functions: -Hold sister chromatids together (through action of cohesin) -Attachment sites for chromosome segregation machinery (through formation of kinetochore, where binds to spindle fibers)

Posttrasnlational modifications

Enzymatic cleavage Addition of chemical constituents may alter the way a protein folds, its ability to interact with other proteins, its stability, its activity, or its location in the cell

X-chromosome inactivation in female mammals occurs through heterochromatin formation

Example of facultative heterochromatin Dosage compensation in mammals so that X-linked genes in XX and XY individuals are expressed at same level Random inactivation of all except one X chromosome in each cell Inactive X chromosome is a Barr body -darkly stained heterochromatin masses observed in somatic cells at interphase

Identification of molecules involved in heterochromatin formation

Genetic screens in Drosophila were used to identify genes involved in chromatin modification -looked for changes in the amount of position-effect variegation -mutations that enhanced heterochromatin formation made eyes more white -mutations that suppressed heterochromatin formation made eyes less white Some genes encode proteins that localize heterochromatin Barrier insulators: block the spread of heterochromatin

Detecting chromosome rearrangements by sequencing

Genome sequencing: -more reads of duplicated sequence in heterozygote -fewer reads of deleted sequence in heterozygote -also, all rearrangement types will juxtapose sequences that are not normally next to each other Amplifying breakpoint by PCR and sequencing: -for following transmission of known breakpoint -inexpensive and sensitive

Rare dominant loss-of-function alleles

Haploinsufficiency: phenotype is sensitive to gene dosage, rare situations in which one wild-type allele does not provide enough of a gene product to avoid a mutant phenotype (i.e. 50% of gene product) ex: GLI3/GLI3+ humans have polydactyly

Chromosomal packaging and function

Heterochromatin: highly condensed, usually inactive transcriptionally -darkly stained regions of chromosomes -Constitutive: condensed in all cells (e.g. most of the Y chromosome and all pericentromeric regons); contain SSRs and transposable elements -Facultative: condensed in only some cells and relaxed in other cells (e.g. position effect variegation, X chromosome in female mammals) Euchromatin: relaxed, usually active transcriptionally -lightly stained regions of chromosomes

Histone tail modifications alter chromatin structure: Acetylation

Histone acetyltransferases (HATs) add acetyl groups to histone tails Prevents close packing of nucleosomes, resulting in open chromatin Favors expression of genes in euchromatin The process is reversed by histone deacetylases, resulting in closed chromatin and repressed transcription

Histone tail modifications alter chromatin structure: Methylation

Histone methyltransferases (HMTases) add methyl groups to histone tails Affect depends on specific amino acid modified Adding methyl group to H3 lysine 9 favors heterochromatin formation The process is reversed by methyltransferases, histone demethylases

Histone proteins

Histones: small, positively charged, and highly conserved -bind to and neutralize negatively charged DNA -five types - H1, H2A, H2B, H3, and H4 -core histones, form the core of most rudimentary DNA packaging unit (H2A, H2B, H3, and H4) make up the nucleosome; H1 acts as a seal to keep core from unwrapping

Summary of phenotypic and genetic effects of deletions

Homozygosity for deletions is often lethal or harmful -depends on size of deletions and affected genes Deletion heterozygotes: -can have a mutant phenotype due to gene dosage effects (haploinsufficiency); half of the normal gene dosage does not produce enough protein product for a normal phenotype -increases risk of phenotype due to mutation in the other copy of the gene, ex: retinoblastoma (tumor suppresor gene) -May uncover existing recessive mutant alleles

Nonhistone proteins

Hundreds of other proteins that make up chromatin and are not histones 200 - 200,000 molecules of each kind of nonhistone protein Large variety of functions: -structural role - chromosome scaffold -chromosome replication, ex: DNA polymerases -chromosome segregation, ex: kinetochore proteins (spindle fibers) -active in transcription - largest group

Telomerase activity and cell proliferation

In humans, the levels of telomerase and cellular life-span varies between different types of cells -Most somatic cells have low expression of telomerase -telomeres shorten slightly at each cell division -senescence after <50 generations in culture -Germ cells, stem cells, and tumor cells have high expression of telomerase -at each generation, telomere length is maintained

X chromosome mosaicism

In very early embryo, both X chromosomes are active In humans, random X-inactivation occurs ~ 2 weeks after fertilization -some cells have maternal X inactivated, other cells have paternal X inactivated -all cell descendants have the same inactive X Adult females are mosaic at X-linked genes

Incompletely dominant loss-of-function

Incomplete dominance: phenotype varies continuously with the amount of functional gene product

Mechanism of translation

Initiation: sets the stage for polypeptide synthesis Elongation: amino acids are added to a growing polypeptide Termination: brings polypeptide synthesis to a halt and enables the ribosome to release a completed chain of amino acids

The karyotype of a human female examined by high-resolution G-banding

Metaphase chromosomes stained with Giemsa have alternating bands of light and dark staining Each band contains many DNA loops and ranges from 1 to 19 Mb in length Banding patterns on each chromosome are highly reproducible darker staining, means more compact DNA, and is inactive light staining, where genes are expressed, transcribed - active chromatin

Phenotypic effects of inversions

Most inversions do not result in an abnormal phenotype Abnormal phenotypes can occur if: -inversion disrupts a gene -inversion places a gene near regulatory sequences for other genes or near heterochromatin Inversions can act as crossover suppressors In inversion heterozygotes, no viable offspring are produced that carry chromosomes resulting from recombination in inverted region

Mutations in genes encoding the components of gene expression have global effects

Mutations in genes encoding gene products for transcription, RNA processing, translation, and protein processing are often lethal Some mutations in tRNA genes can suppress mutations in protein-coding genes noncoding genes: genes for all the rRNAs, tRNAs, and snRNAs that are transcribed but not translated

Structure of tRNA

Ncleotide sequence is the primary structure Secondary structure: (cloverleaf shape) is formed because of short complementary sequences within the tRNA Tertiary Structure: (L shape) is formed by 3-dimensional folding At one end of the L, the tRNA carries an anticodon

Neomorphic alleles

Neomorphic mutations: rare class of of dominant gain-of-function alleles that generate a novel phenotype; generate gene product with new function or that is expressed at inappropriate time or place (ectopic expression) Mutation in Antennapedia gene of Drosophila causes ectopic expression of a leg-determining gene in structures that normally produce antennae ex: Huntington disease

Summary of phenotypic and genetic effects of duplications

Novel phenotypes may occur because of increased gene copy number or because of altered expression in new chromosomal environment Homozygosity or heterozygosity for a duplication can be lethal or harmful -depends on size of duplication and affected genes Unequal crossing-over between duplicated regions on homologous chromosomes can result in increased and decreased copy number

The nucleosome is the fundamental unit of chromosomal packaging

Nucleosomes resemble beads on a string (20A string is DNA) -each bead is a nucleosome with roughly 160bp wrapped around 8 histones (2 of each in core) Linker DNA: ~40 bp that connect one nucleosome with the next H1 outside the core Spacing and structure of nucleosomes affect genetic function -accessibility for proteins that initiate transcription, replication and further compaction -arrangement along chromatin is highly defined and varies in different cell types and under different conditions

Recessive loss-of-function alleles

Null (amorphic) mutations: completely block the function of a protein or gene product (ex: deletion of an entire gene) -ex: heterozygote fulfilled by one allele, loss of the other Hypomorphic mutation: produces either less of the wild-type protein or a mutant protein that functions less effectively than the wild-type protein -gene product has weak, but detectable, activity

Chromosome breakage can produce inversions (In)

Pericentric inversion: centromere is within the inverted segment Paracentric inversion: centromere is not within the inverted segment

Antimorphic (dominant negative) alleles

Prevent the normal protein from functioning Usually occurs in genes that encode multimeric proteins mutant alleles of genes encode proteins that not only fail to provide the activity of the wild-type protein, but also prevent the normal protein from functioning. ex: kinky allele of the axin gene in mice, resulting in malformed (kinky) tail Mutant subunits block the activity of normal subunits

In eukaryotes, the nuclear membrane prevents the coupling of transcription and translation

Prokaryotes transcription and translation take place in cytoplasm and can occur at the same time (coupling) because mRNA extends 5' to 3' direction the same direction as ribosomes move along mRNA. ribosomes can begin to translate a partial mRNA that the RNA polymerase is still in the process of transcribing from DNA -coupling has consequences for regulation of gene expression in prokaryotes No coupling in eukaryotes because the nuclear envelope separates the sites of transcription and RNA processing in the nucleus from the site of translation in the cytoplasm. Translation can affect the rate at which genes are transcribed in more indirect ways

Translation initiation differs in prokaryotes and eukaryotes

Prokaryotes: translation begins at a ribosome binding site on the mRNA, defined by a short sequence of nucleotides called a Shine-Dalgarno box adjacent to an initiating AUG codon. -Polycistronic: they contain the information of several genes (sometimes referred to as cistrons), each of which can be translated independently starting at its own ribosome binding site Eukaryotes: the small ribosomal subunit first binds to the methylated cap at the 5' end of the mature mRNA and then migrates through the 5' UTR to the initiation site. site is almost always the first AUG codon encountered by the ribosomal subunit as it moves along or scans the mRNA in the 5' to 3' direction -Monocistronic: contains the information for translating only a single kind of polypeptide

Transcription requires changes in chromatin structure

Promoters of inactive genes are hidden in nucleosomes To activate a gene, transcription factors bind to enhancers and recruit chromatin remodeling proteins Promoters are exposed by removing or repositioning nucleosomes When a previous inactive gene prepares for transcription in later step of cellular differentiation, the promoter region is observed to change from a DNase resistant site to a DNase hypersensitive site. transcription factors bind DNA at nearby enhancers and recruit proteins that reorganize the chromatin in the vicinity, the new recruit proteins remove promoter-blocking nucleosomes or reposition them in relation to the gene Remodeling complexes: multisubunit of chromatin modulater that uses the energy of ATP hydrolysis to alter nucleosome positioning

Replication at the ends of chromosomes (telomeres)

RNA primers are removed leaving a unreplicated DNA at the 5' ends because nucleotides can only be added to the 3' end Without special mechanism, DNA would be lost from every new DNA strand at each cell cycle Telomerase provides a countermeasure to this limitation of DNA polymerase

Origins of replication in eukaryotes

Rate of DNA synthesis in human cells ~ 50 nt/sec It would take 800 hours to replicate the human genome if there was only one origin of replication -most mammalian cells have ~10,000 origins -many origins are active at the same time -accessible regions of DNA that are devoid of nucleosomes Replication unit (replicon) - DNA being replicated in both directions one origin; the dna running both ways from one origin of replication to the endpoints, where it merges with DNA from adjoining replication forks Autonomously replicating sequences (ARSs), in yeast origin of replication, can be isolated by ability to permit replication of plasmids in yeast cells

Ribosomes, the site of polypeptide synthesis

Ribosomes facilitate polypeptide synthesis: recognize mRNA features that signal the start of translation, help ensure accurate interpretation of the genetic code by stabilizing the interactions between tRNAs and mRNAs, ribosomes supply the enzymatic activity that links the amino acids in growing polypeptide chain, moves 5' to 3' along an mRNA molecule and expose the mRNA codons in sequence ensuring linear addition of amino acids, help end polypeptide synthesis by dissociating both from the mRNA directing polypeptide construction and from the polypeptide product itself

The radial loop-scaffold model for higher level of compaction

Several nonhistone proteins (NHPs) bind to chromatin every 60 - 100 kb and tether the 300 A fiber into structural loops Other NHPs gather several looks together into daisy-like rosettes Condensins may further condense chromosomes into a compact bundle for mitosis (help condense interphase chromosomes into metaphase chromosomes)

Telomeres maintain chromosome integrity

Shelterin proteins bind to telomerase and fold the DNA; folds up the telomeres into a structure that shields single-stranded TTAGGG sequences from nucleases and NHEJ enzymes -protect against degradation by nucleases; nucleases can degrade DNA inward from the broken ends -protects against non-homologous end joining (NHEJ); results in having two centromeres and when separated during anaphase, the DNA will rupture

Locations of genes in relation to chromosomal bands

Short arm = p arm Long arm = q arm Within each arm, light and dark bands are numbered consecutively in regions Idiograms: diagrams of the banding patterns Need high magnification to see parts

Types of mutations in the coding sequence of genes

Silent mutation missense mutation nonsense mutation frameshift mutation

In deletion heterozygotes, pseudodominance can "uncover" a recessive mutation

Similar to a complementation test (the uncovering of a recessive mutant phenotype demonstrates a lack of complementation because neither chromosome can supply wild type gene function) pseudodominance - deletions give the impression that the recessive is dominant Examine phenotype of a heterozygote for recessive allele and deletion: -If the phenotype is mutant, the mutant gene must lie inside the deleted region -If the phenotype is wild-type, the mutant gene must lie outside the deleted region

Functional domains of ribosomes

Small subunit: binds to mRNA Large subunit: has peptidyl transferase activity, which catalyzes formation of the peptide bonds joining adjacent amino acids Three distinct tRNA binding areas: aminoacyl (A) site, peptidyl (P) site, exit (E) site rRNA acts as a ribozyme that joins amino acids together

Wobble

Some tRNAs recognize more than one codon wobble: the flexibility in base pairing between the 3' nucleotide in the codon and the 5' nucleotide in the anticodon wobble position: 5' end of the anticodon wobble bases are modified by specific enzymes that act on the tRNA after it has been synthesized by transcription

Fluorescent in situ hybridization can detect large chromosomal rearrangements

Spectral karyotyping with probes specific for two different chromosomes shows a chromosomal translocation Multicolor banding is produced by using FISH probes specific for particular regions of chromosomes

Initiation Stage

Start codon is AUG at 5' end of mRNA In bacteria, initiator tRNA has formylated methionine (fMet) Prokaryotes: ribosome binds to a Shine-Dalgarno box and an AUG; small ribosomal subunit binds, fMet-tRNA positioned in P site, large subunit binds Eukaryotes: small ribosomal subunit binds to 5' cap, then migrates to the first AUG codon, initiator tRNA carries Met (not fMet)

The four core histone tails can be modified with chemical groups

Tails extend outward from nucleosome Enzymes can add chemical groups (methyl groups, phosphate groups, ubiquitin, etc.) Modified tails can alter nucleosomes and bind chromatin modifier proteins

Types of duplications (Dp)

Tandem duplication: the repeated copies lie adjacent to each other, either in the same order or in reverse order Nontandem (dispersed) duplication: the copies of the region are not adjacent to each other and may lie far apart on the same chromosome or in different chromosomes, can also be in same order or reversed order

Telomerase is a ribonucleoprotein that extends telomeres

Telomerase RNA is complementary to telomere repeat sequences Serves as template for addition of new DNA repeat sequences to telomere Additional rounds of telomere elongation occur after telomeres translocate to newly-synthesized end telomeres have 5' TTAGGG 3' repeats, RNA portion of the enzyme has 3' AAUCCC5' serving has the template for the telomere end Only found in germ cells, especially sperm, not found in somatic cells

Telomeres are "caps" that protect the ends of eukaryotic chromosomes

Telomeres consist of specific repetitive sequences and dont contain genes -species-specific sequences -e.g. TTAGGG in humans, TTGGGG in Tetrahymena -250-1500 repeats with variable number between different cell types Prevent chromosome fusions and maintain integrity of chromosomal ends

Ch. 11

The Eukaryotic Chromosome

Eukaryotic promoters are influenced by enhancers

The promoters recognized by RNA polymerase to initiate transcription are affected by two situations not in prokaryotes -first, the stability of RNA polymerase's interaction with the promoter is often affected by enhancer sequences located far from promoter, second, eukaryotic chromosomes are tightly around histone proteins in a DNA/protein complex called chromatin. to be recognized by RNA polymerase, the promoter of a eukaryotic gene must first be unwound from chromatin, clearing the histones from the promoter is another function of enhancers.

Ch. 8.3

Translation from mrna to proten

tRNAs mediate translation of mRNA codons to amino acids

Translation takes place on ribosomes that coordinate movement of transfer RNAs (tRNAs) carrying specific amino acids tRNAs are short single-stranded RNAs of 74-95 nt -each tRNA has an anticodon that is complementary to an mRNA codon -a specific tRNA is covalently coupled to a specific amino acid (charged tRNA) -base pairing between an mRNA codon and an anticodon of a charged tRNA directs amino acid incorporation into a growing polypeptide

Structure of Ribosomes

Two subunits composed of RNA and protein before translation begins, the two subunits exist as separate entities in the cytoplasm. after the start of translation, they come together to reconstitute a complete ribosome prokaryotic (70S, 50 and 30) eukaryotic (80S, 60 and 40)

Unequal crossing-over can increase or decrease copy number

Unequal crossing-over: recombination resulting from such out-of-register pairing, generates gametes containing increases to three and reciprocal decreases to one in the number of copies of the duplicated region

Different levels of chromosome compaction

When stretched out, the DNA in a single cell would be about 6 feet long Compaction allows the DNA to fit in a cell nucleus compaction starts with winding of DNA around histones, forming small nucleosomes. then tight coiling gathers nucleosomes together into higher-order structures. Nucleosome: condenses naked DNA 7-fold to a 100 A fiber Supercoiling: causes additional 6-fold compaction, achieving a 40 to 50-fold condensation relative to naked DNA Radial loop-scaffold: through progressive compaction of 300 A fiber, condenses DNA to rodlike mitotic chromosomes that are 10,000 times more compact than naked DNA

Heterozygosity for deletions and duplications may have phenotypic consequences

With some genes, an abnormal phenotype can be caused by an imbalance in gene dosage The Notch+ gene is extraordinarily dosage sensitive. Notch+is haploinsufficient and duplication heterozygotes have a mutant phenotype

X-inactivation is initiated by expression of the Xist gene

Xist, X inactivation specific transprict: noncoding RNA (ncRNA) -expressed on the inactive X, but not the active X Xist RNA is a large, non-coding, cis-acting regulatory RNA -binds to the X-chromosome that it was expressed from -Initiates histone modifications (mehtlation, deacetylation) that result in heterochromatin formation

Characteristics of centromeres

Yeast centromeres are two conserved 10-15 bp DNA sequences separated by 90 bp The centromeres of higher eukaryotes are larger and more complex: -Consist of tandem repeats of noncoding satellite DNAs -Repeats of 5-300 bp long, can extend over megabases

charged tRNA

a tRNA covalently coupled to its amino acid. the bond between the amino acid and tRNA contains substantial energy that is later used to drive peptide bond formation

Elongation Stage

amino acids are added to growing polypeptide Ribosomes move in 5' to 3' direction along mRNA Addition of amino acids to C-terminus of polypeptide Charged tRNAs ushered into A site by elongation factors

Loss-of-function mutaitons

any mutation inside or outside a coding region that reduces or abolishes protein activity in many ways

Aminoacul-tRNA synthetases

catalyze attachment of tRNAs to specific amino acids each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA enzyme may recognize several different tRNAs specific for that amino acid.

Silent mutations

do not alter the amino acid sequence -Degenerate genetic code - most amino acids have >1 codon Can change a codon into a mutant codon that specifies exactly the same amino acid. majority of silent mutations change the third nucleotide of a codon

Hypermorphic mutations

generates either more normal protein product than the wild-type allele, or a more efficient mutant protein; generate more gene product or the same amount of a more efficient gene product the FGFR3 gene encodes a transmembrane receptor that is usually active only when bound to the FGF hormone Achondroplasia (dwarfism) is caused by a dominant hypermorphic allele of FGFR3. the altered protein is always active the mutant protein is a constitutively active receptor that is activated all the time. the hypermorphic allele is dominant to the wild type allele because the mutant protein remains active and continues to inhibit bone growth even if the normal protein is present

Chromosome requirements

have to have centromere to identify spindle fibers telomere at tips - protect from getting shortened every round of replication origins of replication, the larger the chromosomes the more origins

Gene mapping using deletions to uncover recessive mutations

if the phenotype of an m/Del heterozygote is mutant, the deletion has uncovered the mutated locus and the gene thus lies inside the region of deletion If the trait determined by the gene is wild type, the deletion has not uncovered the recessive allele, and the gene must lie outside the deleted region

Eukaryotic RNAs are processed to form mRNA

introns interrupt eukaryotic, but not prokaryotic, genes such that the splicing of a primary transcript is necessary for eukaryotic gene expression eukaryotes add a methylated cap (5') and poly-A tail (3') ends of the mRNAs

Mutations outside the coding sequence can disrupt gene expression

mutations in promotors make it hard or impossible for RNA polymerase to associate with the promoter diminish or prevent transcription mutations in enhancers that disrupt them from being recognized by transcription factors also diminish the transcription of eukaryotic genes mutations in termination signal can diminish the amount of mRNA produced and the amount of gene product Eukaryotes: changes in splice acceptor sites, splice donor sites, and branch sites can obstruct splicing, the result will be the absence of mature mRNA and no polypeptide develops, or splicing errors can yield aberrantly spliced mRNAs that encode altered forms of the protein. mutations affecting ribosome binding site would lower the affinity of the mRNA for the small ribosomal subunit, this is likely to diminish the efficiency of translation and thus amount of polypeptide product mutations in stop codon would produce longer than normal proteins that might be unstable or nonfucntional

Missense mutations

mutations that change a codon into a mutant codon that specifies a different amino acid -Conservative - chemical properties of mutant amino acid are similar to the original amino acid, may have little or no effect on protein function; ex: aspartic acid (-) -> glutamic (-) -Nonconservative - chemical properties of mutant amino acid are different from original amino acid, more noticeable consequences ex: aspartic (-) -> alanine (uncharged)

A nonsense mutation in a protein-coding gene creates a truncated, nonfunctional protein

nonsense suppressor tRNAs: tRNA-gene mutations that can suppress the effect of a nonsense mutations in other genes

Termination Stage

polypeptide synthesis stops at the 3' end of the reading frame Recognition of nonsense codons polypeptide synthesis halted by release factors release of ribosomes, polypeptide, and mRNA No normal tRNAs carry anticodons for the stop codons (UAA, UAG, UGA) Release factors bind to the stop codons Release of ribosomal subunits, mRNA, and polypeptide

Differences in eukaryotes and prokaryotes in tanscription and translation

presence of a nuclear membrane in eukaryotes eukaryotic-specific complexities in the mechanisms by which RRNA polymerase recognizes promoters to start transcription variations in the way in which translation is initiated the need for additional transcript processing in eukaryotes

Spectral karyotyping (SKY) is a variation of FISH

probes specific for each chromosome are labeled with a different fluorescent dye SKY: the probes are made from multiple DNAs that originated from positions scattered along the length of individual chromosomes

Translocation

process by which the sequence of nucleotides in a messenger RNA directs the assembly of the correct sequence of amino acids in the corresponding polypeptide Takes place on ribosomes that coordinate the movements of transfer RNAs carrying specific amino acids with the genetic instructions of an mRNA

zymogens

proteins synthesized in inactive forms that are activated by enzymatic cleavage that removes an N-terminal prosegment

Gain-of-function alleles

rare mutations that either enhance a protein's function, confer a new activity on a protein, or express a protein at the wrong time or place usually dominant Hypermorphic mutations Neomorphic mutations Antimorphic alleles

Frameshift mutations

result from insertion or deletion of nucleotides with the coding region -no frameshift if multiples of three are inserted or deleted cause unrelated amino acids or premature stop codons to appear in place of amino acids critical to protein activity, destroying, or diminishing polypeptide function

Polyribosomes consist of several ribosomes translating the same mRNA

simultaneous synthesis of many copies of a polypeptide from a single mRNA

The tRNA anticodon base pairs with an mRNA codon

the amino acid attached to the tRNA will be attached to the growing polypeptide

polyprotein

the larger polypeptide made before it is cleaved into smaller polypeptides

anticodon

three nucleotides complementary to an mRNA codon specifying the amino acid carried by the tRNA always available for base pairing with its complementary mRNA codon anticodon and codon run antiparallel to each other

Position effect variegation (PEV) is Drosophila

white+ (w+) gene is normally located in euchromatin Chromosomal inversion can result in w+ gene being located adjacent to herterochromatin PEV: rearrangements silence w+ gene expression in some cells and not others Red pigment - w+ gene is expressed in all cells No pigment/red pigment - w+ gene is silenced in some cells (none) and expressed in other cells (red) Gene silencing can be caused by spreading of heterochromatin into nearby genes Spreading can occur over >1000 kb of chromatin Heterochromatin spreads further in some cells than in others