Biology 172 Exam 3 University of Michigan

cell division

Process by which a cell divides into two new daughter cells

cell cycle

An ordered sequence of events (including interphase [non-dividing] and the m-phase [dividing]) that extends from the time a eukaryotic cell is first formed from a dividing parent cell until its own division into two cells. The lengths of each stage of the cell cycle can vary in different tissues or organisms

interphase

Cell grows, performs its normal functions, and prepares for division; consists of G1, S, and G2 phases

mitosis

A process of nuclear division in eukaryotic cells conventionally divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase. Conserves chromosome number by equally allocating replicated chromosomes to each of the daughter nuclei.

G1 phase

stage of interphase in which cell grows and performs its normal functions

G2 phase

Cell grows and preps for mitosis

S phase

During ___ of interphase, DNA is replicated.

sister chromatids

identical copies of a chromosome; full sets of these are created during the S(DNA replication) subphase of interphase

centromere

Area where the chromatids of a chromosome are attached

prophase

chromosomes condense

spindle apparatus begins to form

prometaphase

nuclear envelope breaks down

microtubules contact chromosomes at kinetochores

metaphase

Chromosomes line up at the metaphase plate

Chromosomes are being pulled in opposite directions with equal force by the microtubules

Anaphase

sister chromatids separate and are pulled to opposite poles of the cells

telophase

chromosomes decondense

nuclear envelope reforms

cytokinesis

cytoplasm is divided, two daughter cells form

In animals: a ring of actin and myosin filaments contracts inside the cell membrane, causing it to pinch inward in a cleavage furrow

cleavage furrow

The area of the cell membrane that pinches in and eventually separates the dividing cell

chromatin

Clusters of DNA, RNA, and proteins in the nucleus of a cell.

DNA-associated proteins (histones) tightly package DNA into nucleosomes

spindle fibers

Microtubules that move chromosomes during cell division.

kinetochore

A specialized region on the centromere that links each sister chromatid to the mitotic spindle.

metaphase plate

An imaginary plane during metaphase in which the centromeres of all the duplicated chromosomes are located midway between the two poles

homologous chromosomes

Chromosomes that have the same sequence of genes and the same structure

operon

A segment of DNA containing adjacent genes including structural genes and an operator gene and a regulatory gene.

Portion of DNA including a set of genes involved in a specific metabolic pathway

promoter gene

the noncoding sequence of DNA that serves as the initial binding site for RNA polymerase

operator

A specific DNA nucleotide sequence where transcriptional regulatory proteins can bind.

Repressor binds here

regulatory gene

A gene that codes for a protein, such as a repressor, that controls the transcription of another gene or group of genes.

inducer

A specific small molecule that inactivates the repressor in an operon.

polymerase chain reaction

A method of producing thousands of copies of DNA segment using the enzyme DNA polymerase

Steps of PCR

1) Denatuaration

2) Annealing

3) Extension

Denaturation

a solution containing double-stranded template DNA is heated to separate the DNA into two individual strands

Annealing

the solution is cooled, and the two primers anneal to their complementary sequence on the DNA template strands. Hydrogen bonds are made with the template and primer

Extension

DNA polymerase synthesizes new DNA strands by extending the primers

target sequence

the piece of a DNA fragment you are interested in replicatiing

Doubles

each round of amplification _______ the number of molecules that have the same sequence as the template duplex

Keys to PCR

Need to have sequence information to design primers

Heat stable DNA polymerase

PCR components

template DNA

primers

abundant supply of the four dNTPs

Taq DNA polymerse

Buffer (keeps pH of enzyme happy)

plasmid

A small ring of DNA that carries accessory genes separate from those of the bacterial chromosome. Naturally occur in bacteria.

plasmids contain:

an origin of replication

an antibiotic resistance gene (e.g. ampicillin resistance)

a cloning site to insert foreign DNA (multiple cloning site)

Standard Cloning

1) create recombinant plasmid

2) transform plasmid into bacteria

3) Bacterium will make copies of plasmid which makes more copies of plasmid in each cell & more cells

Step 1 of Cloning

cutting and pasting DNA

scissors = restriction enzymes

glue= ligase

Restriction enzymes

endonucleases that cut DNA at specific sequences.

Hundreds of restriction enzymes, each cut at a different sequence

Palindrome

a sequence of DNA that reads the same in either direction

sticky end

Single stranded ends of DNA left after cutting with enzymes

blunt end

Restriction fragments with no overlapping ends and that never combine with another type of DNA

step 2 of cloning

transform plasmid into bacteria

genetic transformation

genetic alteration in a cell as a result to uptake & expression of foreign genetic material

lab technique for efficient transformation

to increase permeability of bacterial cell membrane by chemical treatment or electrical shock

step 3 of cloning

'grow' more plasmid

small

a very _____ fraction of the treated bacterial cells will pick up a plasmid (become transformed)

antibiotics

only cells that have picked up a plasmid can grow in the presence of ____________.

Steps to express human growth hormone in E. coli

Get rid of all intron sequences from the GH1 coding sequence

Add bacterial transcription start (promoter) & transcription termination sequences

Add bacterial ribosome binding site sequence

telomerase

can reverse transcribe

DNA sequencing

used to determine the sequence of an unknown stretch of DNA, nucelotide by nucleotide

Components of DNA sequencing

template DNA

primer

DNA polymerase

deoxynucleotides

dideoxynucleotides

dideoxynuceotide

lacks that 3' hydroxyl group, and it cannot be elongated by DNA polymerase

deoxynucleotide

has a hydroxyl (-OH) group on the 3' carbon, allowing this end to be englongated

single

a _____ sequence reaction will yield few hundred-thousand base pairs of sequence

shotgun sequencing

1) cut genome into small fragments. Small compared to primer. Large compared to genome

2) Sequence each fragment

3) Assemble sequence

open reading frame

A sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon and ending with a termination codon.

introns

there are no _______ in bacteria and achaea, and genes can be identified by highly conserved promoter sequences associated with a distinct ORF

eukaryotic organisms

in _________ _________, genes contain introns, and identification of ORFs is more difficult. One strategy is to use cDNA sequences and then locate the gene in the genome sequence

more conserved

exon

less conserved

intron

no relationship

between genome size & organismal complexity

C-value paradox explanations

Polyploidy. More than 2 copies of their gene

Noncoding DNA

2.5% 50%

protein-coding sequences constitute about ___ of the human genome, & repeated sequences make up more than ___.

90%

In prokaryotic cells, usually ___ of the DNA sequences code for a product used by the cell

Repetitive sequences in eukaryotes

1) transposable elements (about 45% of the genome)

2) short tandem repeats (about 3% of the genome)

transposable elements

are parasitic segments of DNA that are capable of moving from one location in a genome to another

short tandem repeats

microsatellites, or simple sequence repeats, are repeating sequencing of 1 to 5 bases.

minisatellites

variable number terminal repeats (VNTRs), are repeating sequences of 6 to 500 bases

hypervariable

repeat sequences are ___________ (they vary among individuals), and each individual has a unique set

gene expression

process by which a gene produces its product and the product carries out its function

constitutive expression

gene product made continuously

regulated expression

gene product made on demand; expression can be induced or repressed

induced

turning on a gene that is normally off

repressed

turning off a gene that is normally on

transcription

regulated gene expression at the level of __________ is efficient, but can be slow

posttranslational

______________ regulated gene expression is fast, but wastes energy

sugars

E. coli uses _______ for metabolism

glucose

______ is the preferred source of carbon for E. coli because it has a quick breakdown

lactose

if glucose is not available, bacteria can break down _______ to generate glucose

induces

lactose _______ expression of beta-galactosidase

beta-galactosidase

glucose inhibits production of ____-_____________

technique used for gene discovery

genetic screen

find mutant E.coli that has a defect in a gene necessary for metabolism of lactose

genes E. coli needs to utilize lactose

lacZ (encodes beta-galactosidase)

lacY (encodes galactoside permease)

lacZ and lacY

are transcribed as a single polycistronic mRNA

second class of mutants

this strain expresses lacZ and lacY even in the absence of lactose

E. coli turns genes on and off by

DNA binding proteins binding to specific DNA sequences act as switches

negative regulation

repressor bound to DNA & gene gets turned off

positive regulation

activator bound to DNA & gene is turned on

allosteric regulation of LacI

lactose binding changes LacI conformation so that it can no longer bind DNA.

Lactose is an inducer

single regulatory region

operator + promoter

polycistronic RNA

Single bacterial RNA molecule that encodes more than one polypeptide chain; uncommon in eukaryotes.

repressor

product of a separate regulatory gene.

bind the operator and blocks RNA polymerase

catabolite reaction

product of reaction inhibits the enzyme

low

when glucose levels are high, cAMP levels are ___

cAMP

cyclic nucleotide with one phosphate group

CAP

cAMP receptor protein (CRP)

binds cAMP

cAMP-CRP

acts as an activator of the operon

occurs

when there is positive regulation, transcription _______. Helping RNA polymerase. Changing affinity