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Exam 3 Flashcards

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1
Q

What does it mean to say that the code is degenerate?

A

It means that some amino acids are encoded by more than one codon

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2
Q

What does it mean to say that the code is universal?

A

It means that the genes involved in translation are evolutionarily conserved among all organisms

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3
Q

What is the function of ribosomes?

A

Catalyze protein synthesis

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4
Q

What is the function of tRNAs?

A

“Translate” the genetic code from mRNA into an amino acid sequence

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5
Q

What can be said of functional RNA? Why?

A

They make up the largest fraction of cellular RNA at 95% due to their increased stability over mRNA and their high transcriptional activity

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6
Q

What is the function of tRNA synthetases and what is an example?

A

Attach specific amino acids to specific tRNA like glycine-tRNA synthetase catalyzes charging of glycine-tRNA

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7
Q

What is the process of translation initiation?

A

IF3 blocks binding of large to small ribosomal subunit, allowing mRNA to bind to the small subunit; pairing between Shine-Delgarno sequence in mRNA and complementary sequence in 16S rRNA positions mRNA for initiation; IF2 binds GTP and met-tRNA and brings them into the P site; Finally, large subunit associates
with the complex, hydrolyses GTP (releasing energy), and releases IF2 and IF3

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8
Q

What is the process of translation elongation?

A

EF-Tu escorts the charged tRNA into the A site; EF-Ts catalyze release of EF-Tu and GDP from the ribosome; peptidyl transferase catalyzes the formation of peptide bond between amino acids in A and P sites; EF-G catalyzes release of uncharged tRNA and the movement of the ribosome one codon further down the mRNA

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9
Q

What is the process of translation termination?

A

Release factors recognize stop codons in the A site, discharge polypeptide chain, and dissociate ribosomal subunits (RF1 recognizes UAA and UAG and RF2 recognizes UAA and UGA)

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10
Q

What is the wobble effect?

A

The third nucleotide of an anticodon on the 5’ end can form either H bonds with its complementary nucleotide in the 3rd position of the codon or with a different nucleotide in that position

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11
Q

What is a peptide bond?

A

Covalent bonds that link amino acids by linking the amino end of one acid to the carboxyl end of another acid

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12
Q

What happens during formation of a peptide bond?

A

One water molecule is removed

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13
Q

What is a primary protein?

A

Linear sequence of amino acids

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14
Q

What is a secondary protein?

A

Local regions of polypeptides are folded into shapes (helices, beta pleated sheet, zinc finger, etc.)

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15
Q

What is a tertiary protein?

A

Spatial arrangement of amino acids and secondary structures that are farther apart in the linear sequence

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16
Q

What is a quaternary protein?

A

2 or more folded polypeptides (hemoglobin)

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17
Q

What is an active site?

A

Pocket in the enzyme where the substrate binds

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18
Q

What is the formation of a peptide bond described as?

A

A ribosome-catalyzed dehydration reaction

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19
Q

When comparing the polarity of mRNA to the primary amino acid sequence, it can be said that?

A

The 5’ end of mRNA corresponds to the amino terminus of the protein and the 3’ end of mRNA corresponds to the carboxy terminus of the protein

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20
Q

A tRNA with an anticodon 3’ - ACC - 5’ would carry the amino acid…

A

Tryptophan

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21
Q

What are the steps of DNA recombination?

A

Isolate target DNA, cut DNA, ligate target DNA into vector

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22
Q

What is a clone?

A

Genetic copy of an entire organism or an individual gene

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23
Q

What is a vector?

A

Carrier of DNA molecule of interest such as a plasmid, phage, or cosmid

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24
Q

What is an insert?

A

DNA molecule of interest that is a genomic DNA fragment, protein coding sequence, or PCR product

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25
Q

What are some features of vectors?

A

Has multiple cloning site, selectable marker, and origin of replication

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26
Q

What is a multiple cloning site?

A

Multiple restriction sites clustered together in a short stretch of vector DNA

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27
Q

What is a selectable marker?

A

A phenotype test used to select for plasmids with DNA inserts

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28
Q

What is origin of replication?

A

Required for plasmid replication in bacteria in order to start replicating DNA

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29
Q

What are restriction enzymes or endonucleases?

A

Enzymes that cut DNA by recognizing specific DNA sequences and produce sticky or blunt ends

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30
Q

What specific DNA sequences do restriction enzymes recognize?

A

Sites are usually palindromic, recognize 4-8 base pairs, and the enzyme binds to the DNA as a dimer

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31
Q

What is DNA ligase?

A

Enzyme used to covalently link DNA fragments back together to produce recombinant plasmids

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32
Q

Where is the sticky end EcoR1 cut?

A

5’-G/AATTC-3’

3’-CTTAA/G-5’ so there’s a 5’ overhang

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33
Q

Where is the sticky end Pst1?

A

5’-CTGCA/G-3’

3’-G/ACGTC-5’ so there’s a 3’ overhang

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34
Q

What is the blunt end Stu1?

A

5’-AGG/CCT-3’

3’-TCC/GGA-5’

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35
Q

What is antibiotic selection?

A

Antibiotics used to select for bacteria containing plasmids

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36
Q

What is the process of making a genomic DNA library?

A

Isolate genomic dNA, digest genomic DNA with restriction enzymes, ligate digested genomic DNA into vector, and transform vector into bacteria

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37
Q

What is the process of making a cDNA library?

A

RNA is purified from tissue or cell lines, mRNA is purified from rRNA and tRNA by using a column with oligodT to bind the polyA tail, mRNA treated with reverse transcriptase, treated Taq polymerase used to make a second strand, cDNA inserted into cloning vector, cDNA ligated to vector, and vector transformed into bacteria

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38
Q

What is PCR?

A

A series of in vitro reactions to synthesize billions of copies of a specific DNA target

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39
Q

What does Taq polymerase do in PCR?

A

Adds dNTPs

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40
Q

What are all four dNTPs?

A

Building blocks in new DNA strands

41
Q

What do primers do in PCR?

A

Marks starting point within template DNA for DNA polymerase

42
Q

What are the steps of PCR?

A

Target DNA heated to separate strands, temperature lowered, allowing annealing of RNA primers to the ends of target sequence, primers are starting point for synthesis of DNA complementary to template, and process is repeated to amplify DNA

43
Q

What are probes?

A

Find and mark a desired clone because it’s a complementary sequence and can recognize a specific nucleic acid sequence or recognize specific protein

44
Q

What is functional complementation?

A

Use of a cloned fragment of wild-type DNA to transform a mutant into a wild-type

45
Q

What is gel electrophoresis?

A

A method of molecular separation in which DNA, RNA, or proteins are separated in a gel matrix by size, with an electric field drawing molecules through the gel from negative to positive

46
Q

What is southern blotting? Northern blotting? Western blotting?

A

Transfer of electrophoically separated fragments of DNA to an absorbent sheet from a gel that is then immersed in a solution containing a labeled probe that binds to a fragment of interest; same process with RNA; same process with amino acid sequences in proteins

47
Q

What is Sanger sequencing?

A

Amplified DNA can be sequenced where 4 reaction mixtures are set up, each one including: DNA to be sequenced, DNA polymerase, nucleotides, and a labeled chain-terminating variant of 1 of the bases; Polymerase incorporates a chain-terminating variant at random eventually ending the chain at every nucleotide position; the products are run on an electrophoresis gel where the sequence is deduced by reading smallest-largest

48
Q

What is reverse genetics?

A

Start by selecting gene to study, use recombinant DNA techniques to mutate gene, and examine the effect of gene mutation on phenotype

49
Q

What is forward genetics?

A

First select a phenotype of interest, randomly introduce mutations in your population, screen the mutant population for the phenotype, and identify the gene responsible for the mutant phenotype

50
Q

How does PCR-directed mutagenesis work?

A

When you want to change an amino acid you amplify the DNA with a mutant primer where there is a mismatch in the primer on the template that isn’t methylated; transform bacteria; methylated DNA is digested leaving only mutant cDNA

51
Q

How does deletion mutagenesis work?

A

You produce a series of deletion mutants of cDNA and transfect mutant and wild-type that was inserted into a vector into a eukaryotic cell line; add cortisol to see if the mutant can still bind the hormone and measure via the reporter gene (usually green fluorescent protein)

52
Q

What are the findings of deletion mutagenesis?

A

The mutants cannot bind to some important functional domains

53
Q

What is the zinc finger domain important for?

A

DNA binding

54
Q

What is gene replacement?

A

Substituting a mutant allele for a normal allele

55
Q

What is gene knockout?

A

Insertion of non-functional allele for a gene

56
Q

How is a target vector made?

A

Some sequences homologous to target gene, positive selection marker (neomycin) within target sequence, negative selection (tk) outside target sequence

57
Q

You wish to probe a human cDNA library to find out which insert has the beta-globin gene. What would be a good choice as your probe?

A

A 60 nucleotide probe corresponding to exon 1 for beta-globin

58
Q

True or False? Taq polymerase copies the sequence of mRNA into the first strand of DNA.

A

False; second

59
Q

True or False? Just like PCR, Sanger dideoxy DNA sequencing uses two primers in each reaction.

A

False

60
Q

A cDNA library is produced from what and is valued in the analysis of what?

A

A DNA copy of mRNA isolated from a group of cells; the protein-encoding open reading frame of a gene (no introns)

61
Q

What is the process of going from genomes to phenotype?

A

Genomics, transcriptomics, proteomics, metabolomics, and phenotype

62
Q

What is genomics responsible for?

A

What responses are possible in genes

63
Q

What is transcriptomics responsible for?

A

Initiation of response

64
Q

What is proteomics responsible for?

A

It’s what drives the response

65
Q

What is metabolomics responsible for?

A

The actual response and display of the phenotype

66
Q

What is new in medical genomics and how is it used?

A

Personal genomes that can diagnose diseases and predict susceptibility and allow us to develop new ways to treat, cure, or prevent diseases

67
Q

What is ecological genomics?

A

Investigation of organismal responses to environmental changes at the molecular genetics level

68
Q

What is metagenomics?

A

Rapid sequencing of genomes of a mixture of organisms directly from environmental samples

69
Q

What is the reason for metagenomics?

A

Allows for discovery of new microbes, monitoring impact of pollutants on ecosystems, studying microbial communities in extreme environments, and identifying new genes for use in medicine, agrochemicals, pharmaceuticals, and more

70
Q

What is structural genomics?

A

Molecular organization of the entire genome

71
Q

What does structural genomics allow us to do?

A

Sequence the genome to produce a high resolution DNA sequence for each chromosome, create physical maps of large cloned DNA fragments, assign genes to chromosomes via FISH, and locate new genes

72
Q

What are the two ways to complete a genomic library?

A

Map-based sequencing and whole-genome shotgun sequencing

73
Q

What are contigs?

A

Groups of overlapping pieces of chromosomal DNA that allow us to make contiguous clones

74
Q

What are the steps in map-based sequencing?

A

Use molecular markers to make a physical map of the large cloned fragments, compare restriction enzyme digest patterns among BACs, BACs from the same genome region that overlap will share some of the same restriction enzyme sites, select minimum tiling path, divide into subclasses, and assemble the fragments to create the genome

75
Q

What are the steps in whole-genome shotgun sequencing?

A

Mechanical shearing or restriction digest, construction of several libraries, vector inserts of individual bacteria are sequenced, smaller DNA fragments assembled into larger DNA fragments by finding overlapping sequences, assembly into contigs of overlapping sequences

76
Q

Why is whole-genome shotgun sequencing hard for larger genomes especially?

A

It’s hard due to repetitive DNA such as the sequence “CAT”

77
Q

What solves the problem of whole-genome sequencing?

A

Paired-end sequence reads

78
Q

How do we find the sequences in a genome?

A

Computational searches for open reading frames based on similarity to known genes from the same species, use expressed sequence tags (ESTs) and full-length cDNAs to find eons, untranslated regions, and start and stop codons, search for regulatory sequences based on known consensus motifs, and use comparative genomics to find similar genes from other species

79
Q

What is comparative genomics?

A

Using evolution to make a large-scale comparison across species of genomes

80
Q

What are some assumptions made for comparative genomics?

A

Biology is shared by different species, analyzing multiple species together increases information, and we can interpret gene function in organism of study by studying orthologs in other, possibly simpler organisms

81
Q

What is synteny?

A

Large linkage groups are evolutionary conserved

82
Q

What is true of all organisms?

A

We all come from a common ancestral genome

83
Q

What can we learn when comparing DNA sequences between diverse species?

A

We can identify essential elements that are evolutionarily conserved like gene coding regions (i.e. intron-exon boundaries) and regulatory regions (i.e. transcription factor binding sites)

84
Q

What are homologs?

A

A gene related to a second gene by descent from a common ancestral DNA sequence by either speciation or gene duplication

85
Q

What are orthologs?

A

A type of homolog that are genes in different species that have evolved from a common ancestral gene via speciation that retain the same functions in the course of evolution

86
Q

What are paralogs?

A

Type of homolog that are genes produced via gene duplication within a genome that typically evolve new functions or become pseudogenes

87
Q

What is a transcriptome?

A

The sequence and expression patterns of all transcripts

88
Q

What is a proteome?

A

The sequence and expression patterns of all proteins

89
Q

What is yeast two-hybrid?

A

Assay for interactions between proteins

90
Q

What do microarrays do?

A

Use complementary DNA to measure levels of mRNA

91
Q

What are oligonucleotide arrays?

A

Small sequence-specific strands of DNA are synthesized on glass slides in an ordered array

92
Q

What are cDNA arrays?

A

cDNAs for particular genes are attached to glass slides in an ordered array

93
Q

What is the process of completing a microarray?

A

The probes are on a slide and the RNA is hybridized to the probes

94
Q

What are the steps of cDNA array synthesis?

A

Grow bacteria with plasmid carrying cDNA, isolate plasmid carrying cDNA, PCR amplification of cDNA, and spot PCR product on slide using a robot

95
Q

What is the process of two-channel array hybridization?

A

Extract total RNA and purify out mRNA that’s converted to cDNA and incorporate nucleotides with fluorescent tags, group cDNA and cohybridize onto microarray slide, and wash to remove cDNA not bound to the probe and plot the fluorescence intensity

96
Q

What happens if the treatment : control ratio is 1? Greater than 1? Less than 1?

A

There is no difference in gene expression; gene is more abundant in treated sample; gene is more abundant in control sample

97
Q

How does yeast two-hybrid assay work?

A

One gene (bait) is spliced next to the DNA binding domain of GAL4 on one plasmid, another gene (prey) is spliced next to the transactivation domain of GAL4 on another plasmid, the cell is transfected and the GAL4 responsive element forms

98
Q

Studies of the transcriptome, proteome, or interactome are generally referred to as…

A

Functional genomics

99
Q

What bases are involved in the wobble effect?

A

If the 5’ end of the anticodon is G, the 3’ end of the codon can be C or U; If the 5’ end of the anticodon is U, the 3’ end of the codon can be G or A; If the 5’ end of the anticodon is I, the 3’ end of the codon can be U, C, or A

Genetics (5 decks)

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