Lecture 15

Question 1

genome

Answer 1

complete set of DNA in a single cell of an organism

Question 2

genomics

Answer 2

the study of genomes

Question 3

structural genomics

Answer 3

gene organization and components

Question 4

functional genomics

Answer 4

looking at different species; evolution

Question 5

metagenomics

Answer 5

complex enviromental samples

Question 6

what is structural genomics?

Answer 6

• sequencing genomes

- analyzing nucleotide sequences to identify genes and sequences such as gene-regulatory elements

Question 7

protein coding regions

Answer 7

-annotation of sequence reveals several identifiable features indicating that the sequence contains a protein-coding gene

Question 8

examples of protein coding genes

Answer 8

• promoter sequence
• initiation sequence
• three exons two unshaded areas between exons represents introns; later spliced out during RNA processing

Question 9

open reading frames (ORF)

Answer 9

• sequences of triplet nucleotides translated into amino acid sequence of a protein
• suggestive of protein endoding gene
• typically begin with inititation sequence ATG
• ends in a termination sequence TAA, TAG, and TGA

Question 10

what is functional genomics?

Answer 10

• study if gene functions
• based on resulting RNAs
• based on possible proteins they encode and regulatory elements

Question 11

BLAST searches

Answer 11

used to screen databases and compate a sequence to a known sequence

Question 12

similarity searches

Answer 12

• a genome sequence statistically similar to gene with known function likely encodes for protein with similar function
• like comparing protions of the human leptin gene (LEP) with its homolog in mice

Question 13

homologous genes

Answer 13

genes that are evolutionarily related

Question 14

orthologs

Answer 14

genes from different species thought to have decended from a common ancestor

Question 15

paralog

Answer 15

homologous genes in same species

Question 16

protein domains

Answer 16

ion channels, membrane-spanning regions, secretions, and ecport signals

Question 17

motifs

Answer 17

helix-turn-helix, leucine zipper, or zinc-finger motif

Question 18

protein domains and motifs

Answer 18

when a gene sequence is used to predict polypeptide sequence, the polypeptide can be analyzed for specific protein domains and motifs

Question 19

genomic techniques

Answer 19

• range of techniques valuable for functional genomic studies
• like chromarin immunoprecipitation (ChIP)

Question 20

how is ChIP useful?

Answer 20

• designed to map protein-DNA interactions

- useful for identifying genes regulated by DNA-binding protein (DBP) transcription factors

Question 21

what is the ChIP method?

Answer 21

• DNA is crosslinked to the proteins that are bound to the DNA
• DNA is isolated and fragmented with DBPs attatched
• specific DBP is target with an antibody
• antibody is used to purify the DNA DBP complex
• crosslink is disrupted
• DNA is sequenced to reveal the DBP binding site

Question 22

what did the human genome project reveal about the organization of the human genome?

Answer 22

-illustrated that all humans and all other species share a common set of genes essential for cellular function, reproduction, and development

Question 23

alternative splicing

Answer 23

• HGP revealed that the number of genes is lower than the number of predicted proteins
• many genes encode for multiple proteins through alternative splicing
• can generate multiple mRNAs

Question 24

functional categories assigned for human genes on the basis of

Answer 24

• functions determined previously
• comparisions to known genes and predicted protein sequences from other species
• predictions based on annotation and analysis of protein functional domains and motifs

Question 25

single-nucleotide polymorphisms (SNPs)

Answer 25

- single base chnages in genome | - variations associated with disease conditions

Question 26

copy number variations (CNVs)

Answer 26

-segments duplicated or deleted

Question 27

what percentage of the human genome is composed of endogenous retroviruses?

Answer 27

8%

Question 28

CRISPR-Cas

Answer 28

- Clustered Regularly Interspaced Short Palindromic Repeat | - a short, particularly palindromuc repeated DNA sequences found in the genomes of bacteria and other microorganisms

Question 29

what is the CRISPR-Cas mechanism?

Answer 29

- spacer acquisition - crRNA biogenesis (RNA from CRISPR gene) - target interference

Question 30

spacer acquisition

Answer 30

- invading phage DNA is cleaved into smaller fragments known as photospacers, which are then inserted into CRISPR loci to become new spacers - when new spacers are added to the CRISPR locus, repeat sequences are duplicated such that each spacer is flanked on each side

Question 31

crRNA biogenesis

Answer 31

- CRISPR loci are transcribed starting at the promoter within the leader, into long RNA transcripts - processed into short CRISPR-derived RNAs each containing a single spacer flanked on both sides by repeat sequences

Question 32

target interference

Answer 32

- mature aRNAs associate with Cas nucleases, ir nuclease comlexes, and recruit them to complementary sequences in invading phage DNA - Cas nucleases then cleave the viral DNA, thus nutreulizing infection

Question 33

CRISPR-Cas type 1

Answer 33

requires multi-subunit protein complexes to mediate RNA-guided viral DNA destruction during the interference step

Question 34

CRISPR-Cas type 2

Answer 34

-the single Cas9 protein is sufficient, Cas9 also plays a role in spacer aquition and crRNA biogenesis

Question 35

Cas9 during spacer aquisition: cleave invading viral DNA

Answer 35

- Cas9 selects protospacer sequences flanked by a protospacer adjacent motif (P A M) defined by the sequence 5’N G G3’. N = A,G,T or C - After protospacer cleavage, the Cas1/Cas2 complex integrates the D N A into a C R I S P R locus

Question 36

Cas9 during crRNA biogenesis

Answer 36

- Noncoding R N A called a transactivating c r R N A (tracrR N A) binds to c r R N A repeat - Complex is then bound by Cas9 and cleaved into mature c r R N A/ tracrR N A duplexes by an R Nase that specifically recognizes dsR N A (R Nase Ⅲ)

Question 37

CAs9 during target interference

Answer 37

- Cas9 has two nuclease domains that cleave the target D N A sequence - The H N H domain cleaves the strand of viral D N A that is complementary to the c r R N A - The R u v C domain cleaves the noncomplementary strand - Cas9 will only cleave the D N A if the target is adjacent to a P A M sequence

Question 38

what is the difference between type I and type II CRISPR-Cas Systems?

Answer 38

- type I requires multi-subunit protein complexes to mediate RNA-guided viral DNA destruction during the interference step - in type II, the single CAS9 protein is sufficient, Cas9 also plats a role in spacer acquisition and crRNA biogenesis