Cell Biology Exam 2
nested gene
A gene contained within an intron of a larger host gene
What characteristics can you use to identify a gene that has been copied by reverse transcription?
A gene copied by reverse transcription would lack introns
Describe three possible effects of the insertion of a transposable element on a target gene.
A transposable element could 1. increase target gene expression by inserting a new promoter, 2. decrease target gene expression by disrupting the relationship of a target gene to its normal promoter, 3. or inactivate a target gene by inserting within it
transposon
A transposable genetic element that moves within a genome by means of a DNA intermediate.
DNA molecules with different base compositions denature at different temperatures. How would you expect the denaturation temperature of a DNA molecule that's rich in AT base pairs to compare with that of a DNA molecule that's rich in GC base pairs?
AT base pairs have two hydrogen bonds compared to three in GC base pairs. GC base pairs are therefore more stable and a DNA fragment rich in GC pairs would denature at higher temperature.
genome
All the genetic information in an organism; all of an organism's chromosomes.
What was the purpose of ampicillin selection?
Ampicillin is used to select for bacteria that contain the recombinant plasmid. The pBR322 plasmid contains an ampicillin-resistance gene; therefore, only bacteria that were transformed successfully can survive on media containing ampicillin. This type of selection is not able to differentiate between bacteria that contain recombinant plasmids (containing the viral DNA insert) and those that contain "empty" vectors (without the viral DNA insert).
You have made a library in a plasmid vector containing complete human cDNAs. What is the average size of an insert?
Approximately 4.3 kb, the total exon sequence of an average human gene
Why did the bands migrate closer to the cathode as the length of digestion increased?
As the length of digestion was increased, more and more linker DNA was cleaved, resulting in increasing quantities of mononucleosome- containing fragments
What kind of DNA molecules are present in bands a, b, c, and d?
Bands a, b, c and d contain DNA molecules corresponding to mono-, di-, tri-, and tetra nucleosomes, respectively
How do intron sequences in the human genome increase the diversity of proteins expressed from the limited number of approximately 20,000 genes?
By allowing alternative splicing to produce multiple proteins from the same gene
Why do DNA molecules migrate toward the + electrode? What determines the rate of their migration?
DNA migrates toward the + electrode because it is negatively charged, due to the phosphates in its backbone. The length of the DNA fragments determine the rate of their migration. The smaller the fragment, the faster it can move through the gel. At the end of electrophoresis, larger DNA molecules will be found closer to the loading wells, and smaller fragments will have moved to- ward the bottom of the gel.
cDNA
DNA molecule that is complementary to an mRNA molecule, synthesized in vitro by reverse transcription
vector
DNA molecule used to direct the replication of a cloned DNA fragment in a host cell
reverse transcription
DNA polymerase that uses an RNA template
How do these other bands compare with the pattern of BamHI-digested HSV-1 DNA (sample 6)?
Each band comigrates with a corresponding fragment in the HSV-1 DNA digest. This is presumably true for samples 1-4 as well, even though those bands—corresponding to small DNA fragments that bind less fluorescent dye and cannot be visualized—do not show up in the HSV-1 DNA digest (sample 6).
How would RNAi against human exportin-t affect human fibroblasts in culture?
Exportin-t is required for the export of tRNAs from the nucleus, so RNAi against exportin-t would prevent tRNA export and lead to inhibition of translation.
You grow E. coli for several generations in media containing 15N. The cells are then transferred to media containing 14N and grown for two additional generations. What proportion of the DNA isolated from these cells will be heavy, light, or intermediate in density?
Half the DNA will be intermediate density and half will be light.
Why do DNA sequencing reactions contain all four normal nucleotides as well as one dideoxynucleotide?
In order to allow synthesis of DNA fragments of varying length before they are terminated by di deoxynucleotide incorporation.
Nucleic acids have a net negative charge and can be separated by gel electrophoresis on the basis of their size. In contrast, different proteins have different charges. How then can proteins be separated on the basis of size by electrophoresis?
In order to separate proteins by size, they are denatured with the detergent sodium dodecyl sulfate (SDS). Proteins bind many molecules of SDS, giving each protein a negative charge and allowing separation on the basis of size.
An alternative model for DNA replication might be called "conservative replication," in which the two parental strands stay together and give rise to a progeny molecule consisting of two newly synthesized strands. How could you distinguish this experimentally from semiconservative replication?
In the Meselson-Stahl experiment, conservative replication would predict that half of the DNA would be heavy and half would be light after replication for one generation. No hybrid molecules would be formed.
You are studying a gene that is found in euchromatin in brain cells but in heterochromatin in other cell types. What do you infer about its function?
It is probably only transcribed in brain cells
You have isolated a cDNA clone of a gene of interest. How could you predict the amino acid sequence of the protein it encodes
Knowing the genetic code, you can predict the amino acid sequence encoded by the cDNA in all three possible reading frames. It is likely that only one will not be interrupted by a termination codon, and this open reading frame corresponds to the amino acid sequence of the encoded protein.
What roles do lamins play in nuclear structure and function
Lamins form a filamentous network that supports and stabilizes the nuclear envelope. Lamins also provide binding sites for chromatin to attach to the inside of the nuclear envelope. In addition, many proteins that participate in transcription, DNA replication, and chromatin modification interact with lamins.
LINE (long interspersed element)
Member of a family of highly repeated retrotransposons in mammalian genomes.
SINE (short interspersed element)
Member of a family of highly repeated retrotransposons in mammalian genomes.
Approximately how many DNA fragments would be generated by cleavage of yeast DNA with the enzyme Not1?
Not1 has a recognition site of 8 base pairs, so cleavage with Not1 yields fragments with an average size of approximately 66 kb (48 = 65,536). The yeast genome is 12 × 106 base pairs, so Not1 digestion would yield approximately 18 fragments of yeast DNA.
Approximately how many molecules of histone H1 are bound to yeast genomic DNA?
One molecule of histone H1 binds genomic DNA approximately every 200 bp. So the number of histone H` molecules bound to the yeast genome can be determined by dividing the size of the yeast genome (12 Mb) by 200 bp to yield a value of 60,000
Suppose you find that PFL is localized to cell nuclei. What possible functions would this suggest?
Possibilities would include DNA replication or transcription.
How do primers determine the region of DNA that's amplified in PCR?
Primers hybridize to sites on both sides of the region to be amplified by complementary base pairing. They then direct DNA synthesis between them
How can protein-coding sequences be identified in the DNA sequence of a genome?
Protein-coding sequences can be identified as long stretches of nucleotide sequence that do not contain chain-terminating codons.
How can relative levels of expression in the retinas of 1-day-old and 28-day-old mice be determined in this experiment?
Relative levels of expression of each gene represented as a spot on the array are indicated by the color of fluorescence.
What amino acid substitutions would you make in histones to reduce their binding to DNA?
Replacing the basic amino acids lysine and arginine with neutral or acidic amino acids would reduce DNA binding
Why does the undigested sample (sample 1) appears as it does?
Sample 1 contains undigested genomic DNA molecules. Because these are very large molecules. Because these are very large molecules, they can move only a short distance from the well toward the positive electrode
How do simple-sequence repeats differ from transposable elements
Simple-sequence repeats are tandem arrays of short repeated sequences Transposable elements are dispersed throughout the genome
What conclusions can be drawn from observations of spots A, B, C, and D?
Spot A hybridized to similar extents with cDNAs from both 1-day-old and 28-day-old mice, resulting in a yellow spot (red and green together produce a yellow spot). The gene corresponding to spot A is expressed in both 1-day-old and 28-day- old mice. Spot B hybridized only with cDNAs synthesized in 1-day-old mice, resulting in a green spot. The gene corresponding to spot B is expressed only in 1-day-old mice. Spot C did not hybridize to the fluorescent cDNA molecules present in either sample, result- ing in the black spot on the chip. The gene corresponding to spot C is not expressed in retinas of either 1-day-old or 28-day-old mice. Spot D hybridized only with cDNAs synthesized in 28-day-old mice, resulting in a red spot. The gene corresponding to spot D is expressed only in 28-day-old mice.
Explain the ladder-like band patterns in samples 2 to 5.
Staphylococcus nuclease cleaves linker DN molecules between the nucleosomes randomly, so the chromatin fragments it creates separate into ladder-like band patterns in the gel
How are the "sticky ends" produced by restriction endonucleases useful in molecular cloning?
Sticky ends allow DNA fragments to associate by complementary base pairing and be joined by DNA ligase.
How are DNA molecules visualized in a gel after electrophoresis?
The DNA molecules in a gel are visualized by staining them with a fluorescent dye that binds to DNA. Usually, the fluorescent dye is incorporated into the gel during its preparation. After electrophoresis, the DNA molecules appear as fluorescent bands in the gel when exposed to ultraviolet light
How does Ran determine the directionality of nuclear import?
The distribution of Ran/GTP across the nuclear envelope determines the directionality of nuclear transport
You are using RNA-seq to analyze gene expression in breast cancers compared to normal breast cells. One gene of potential interest yields about five times the frequency of reads in normal cells. What does this say about the gene's level of expression in the two cell types? Do you think this gene could have a role in cancer?
The gene is expressed at five times higher level in the cancer cells compared to normal breast cells. It could play a role in the disease.
Starting with DNA from a single sperm, how many copies of a specific gene sequence will be obtained after 10 cycles of PCR amplification? After 30 cycles?
The haploid sperm contains a single starting copy of the DNA sequence. Each cycle of PCR amplifies the starting material twofold, so 10 cycles yields 210, or approximately a thousand, copies. Amplification for 30 cycles yields 230, or more than a billion, copies.
You are interested in a protein called Mox, which is found in the nucleus of cancer cells but the cytoplasm of normal cells by immunofluorescence. Mass spectrometry indicates that one peptide of Mox differs by a molecular mass of 78 in cancer versus normal cells, with the higher- mass peptide found in the nucleus. How do you interpret these results?
The mass of a phosphate group is 78, so it appears that Mox is phosphorylated in cancer cells and that phosphorylation directs its import to the nucleus.
describe the structure of the nucleus
The nucleus is composed of the cell's DNA, pierced with holes called pores, allows large molecules to pass between the nucleus and cytoplasm and it has a nucleolus
What are the other bands?
The other bands are the cloned viral DNA fragments.
semiconservative replication
The process of DNA replication in which the two parental strands separate and serve as templates for the synthesis of new progeny strands
What is the effect of PEG on DNA fragments of different sizes? How is this influenced by the concentration of PEG?
The reference sample (sample 1) contains several different DNA fragments. At 10% PEG (sample 2), the smallest DNA fragments disappear from the gel, indicating that these fragments remain in solution at 10% PEG. As the concentration of PEG decreases, longer and longer DNA frag- ments remain in solution (samples 3 and 4). At 5% and lower PEG concentrations (samples 5-7), no DNA fragments appear on the gel, indicating that all lengths remain in solution. PEG precipi- tates the DNA molecules from solution. The size of DNA fragment precipitated from solution is inversely related to the concentration of PEG in the solution. The smaller the DNA fragment, the higher the concentration of PEG necessary to precipitate it.
How did studies of adenovirus facilitate the discovery of introns?
The small genome of adenoviruses and their robust replication in infected cells allowed the ready analysis of viral DNA and mRNA's
What was the purpose of tetracycline selection?
The tetracycline-resistance gene in the vector contains the BamHI cleavage site; therefore, if viral DNA fragments were ligated into the vector during the cloning process, the gene would be inactivated. Thus, bacteria containing a recombinant plasmid are sensitive to tetracycline treatment.
How does the total amount of protein-coding sequence in the human genome compare to that in Drosophila? How about the total amounts of noncoding sequence?
The total amount of protein-coding sequences in humans is less than twice that in Drosophila. In contrast, the amount of human noncoding sequence is more than 30 times the amount in Drosophila.
What is the average length of an intron in human genes?
We can calculate the average length of a human intron by dividing the total length of intron sequence by the number of introns in an average human gene: approximately 5800 bp
You are studying a protein called Nef, which is phosphorylated in nerve cells but not in other cell types. You hypothesize that phosphorylation affects the participation of Nef in protein-protein interactions. How can you compare the proteins that interact with phosphorylated versus nonphosphorylated Nef? Would the yeast two-hybrid system be useful in this analysis?
You could compare the proteins that are associated with Nef by immunoprecipitation of nerve cells and other cell types. The yeast two-hybrid system would not be useful since Nef wouldn't be phosphorylated in yeast.
You have cloned a cDNA encoding a protein (called PFL) that you think may be involved in cell growth. Describe three experimental approaches you could use to determine where the protein is localized within cells
You could prepare an antibody based on the cDNA sequence or against the encoded protein expressed in bacteria. The antibody could be used to determine protein localization by immunofluorescence or by subcellular fractionation followed by immunoblotting. Alternatively, you could generate a recombinant molecule in which the cDNA is joined to GFP and transfect animal cells to determine protein localization by fluorescence microscopy.
NF-kB
a family of transcription factors that are activated in response to a variety of stimuli; important transcription factor- plays a huge role in a large number of cellular functions
dna microarray
a glass slide or membrane filter onto which oligonucleotides or fragments of cDNA's are printed at a high density, allowing simultaneous analysis of thousands of genes by hybridization of the microarray with fluorescent probes
gene family
a group of related genes that have arisen by duplication of a common ancestor
exportins
a karyopherin that recognizes nuclear export signals and directs transport from the nucleus to the cytosol
importins
a karyopherin that recognizes nuclear localization signals and directs nuclear imports
nuclear pore
a large structure forming a transport channel through the nuclear envelope
miRNA
a naturally occurring short noncoding RNA that acts to regulate gene expression
UTRs (untranslated regions)
a noncoding region at the 3' and 5' end
intron
a noncoding sequence that interrupts exons in a gene
psuedogene
a nonfunctional gene copy
telomerase
a reverse transcriptase that synthesizes telomeric repeat sequences at the ends of chromosomes from its own RNA template
exon
a segment of a gene that is included in a spliced mRNA
snoRNA (small nucleolar RNA)
a small RNA present in the nucleolus that functions in pre-rRNA processing
open reading frames
a stretch of nucleotide sequence that does not contain stop codons and can encode a polypeptide
Retrotransposons
a transposable element that moves via reverse transcription of an RNA intermediate
What are the approximate percentages of each of the following kinds of sequences in the human genome?
a. protein-coding sequence, 1.2%; b. introns, 35%; c. All transcribed sequence, 75%
NLS (nuclear localization signal)
amino acid seq that targets proteins for transportation from the cytoplasm to the nucleus
restriction endonucleases
an enzyme that cleave DNA at specific sequence
lamin
an intermediate filament protein that forms the nuclear lamina
nuclear envelope
barrier separating the nucleus from the cytoplasm, composed of an inner and outer membrane, a nuclear lamina, and nuclear pore complexes
chromosome
carrier of genes, consisting of long DNA molecules and associated protein
karyopherins
carry cargo through the NPC
Heterochromatin
condensed, transcriptionally inactive chromatin
heterochromatin
condensed, transcriptionally inactive chromatin
euchromatin
decondensed, transcriptionally active interphase chromatin
Euchromatin
decondensed, transcriptionally inactive interphase chromatin
RNAi (RNA interference)
degradation of mRNAs by short complementary double-stranded RNA molecule
DNA ligase
enzyme that seals breaks in DNA strands
Gene
functional unit of inheritance, corresponding to a segment of DNA that encodes a polypeptide or RNA molecule
mutation
genetic alteration
genotype
genetic composition of an organism
rna seq
global analysis of RNA's by next-generation sequencing
knockout clones
inactivation of a chromosomal gene by homologous recombination with a cloned mutant gene
How would inactivating a gene encoding a miRNA by CRISPR/Cas affect expression of genes targeted by that miRNA?
increase expression of genes targeted by that miRNA
proetomics
large scale analysis of cell proteins
histone
member of a family of proteins that package DNA in eukaryotic chromosomes
nuclear lamina
meshwork of lamin filaments provided structural support to the nucleus
PCR (polymerase chain reaction)
method for amplifying a region of DNA by repeated cycles of DNA synthesis in vitro
gel electrophoresis
method in which molecules are separated based on their migration in an electric field
How do miRNAs and lncRNAs regulate expression of their target genes?
miRNAs target mRNAs to block translation and induce mRNA degredation, lncRNAs regulate transcription
antigen
molecule against which an antibody is directed
next generation sequencing
new methods that allow rapid sequencing of billions of bases of DNA
The activity of "transforming principle" in the experiments of Avery, MacLeod, and McCarty was abolished by treatment with enzymes that digest DNA but not by enzymes that digest proteins. What effect do you think treatment with enzymes that digest RNA would have
no effect
speckles
nuclear body that contains components of the mRNA splicing machinery
nucleolus
nuclear sight of rRNA transcription, processing, and ribosome assembly
nuclear membrane
one of the membranes forming the nuclear envelope; the outer nuclear membrane is continuous with the ER and the inner nuclear membrane is adjacent to the nuclear lamina
phenotype
physical appearance of an organism
human genome project
project whose goal is to map, sequence, and identify all of the genes in the human genome
antibody
protein- also called immunoglobin- produced by B lymphocytes that binds to a foreign molecule
LADs (lamina associated domain)
region of heterochromatin associated with the nuclear lamina
NADS (nucleolus associated domain)
region of heterochromatin associated with the nucleolus
telomere
repeat of simple- sequence DNA that maintains the ends of linear chromosomes
RAN
small GTP-binding protein involved in nuclear import and export
plasmids
small, circular DNA molecule capable of independent replication in a host cell
origin of replication
specific DNA sequence that serves as a binding site for proteins that initiate replication
genomics
study of whole genomes, including genes and their functions
CRISPR/Cas
system for introducing targeted mutations into mammalian genes consists of CRISPR RNA's that recognize specific target sequences and Cas proteins that cleave the targeted DNA
nucleosome
the basic structural unit of chromatin consisting of DNA wrapped around a histone core
chromatin
the fibrous complex of eukaryotic DNA and histone proteins
molecular cloning
the insertion of a DNA fragment of interest into a DNA molecule (vector) that is capable of independent replication in a host cell
What do the bands indicated by the arrow contain?
the linearized plasmid DNA fragments.
transcriptomes
the synthesis of an RNA molecules from a DNA template
Immunoprecipitation
the use of antibodies to isolate proteins
retrovirus
virus that replicates by making DNA copy of its RNA genome by reverse transcription
