How to find a gene?*

• One way is too search for an open reading frame (ORF).

• An ORF is a sequence of codons in DNA that starts with a Start codon, ends with a Stop codon, and has no other Stop codons inside.

* = inexact science

Each strand has 3 possible ORFs.

5'                                3’ atgcccaagctgaatagcgtagaggggttttcatcatttgagtaa

1 atg ccc aag ctg aat agc gta gag ggg ttt tca tca ttt gag taa M   P   K   L   N   S   V   E   G   F   S   S   F   E   * 

2  tgc cca agc tga ata gcg tag agg ggt ttt cat cat ttg agt  C   P   S   *   I   A   *   R   G   F   H   H   L   S   

3   gcc caa gct gaa tag cgt aga ggg gtt ttc atc att tga gta   A   Q   A   E   *   R   R   G   V   F   I   I   *   V     

Eukaryotic Genomes

• Finding a gene is much more difficult in eukaryotic genomes than in prokaryotic genomes. WHY??

Prokaryotic (bacterial) genomes:

• Are much smaller than eukaryotic genomes-

E. coli = 4,639,221 bp, 4.6 Mb

Human = ~~ 3,300 Mb

Prokaryotic (bacterial) genomes:

• Contain fewer genes:

E. coli- 4285 protein coding genes

- 122 Structural RNA genes

• Human- ~ ~ ~ 32,000 genes

Prokaryotic (bacterial) genomes:

Contain a small amount of noncoding DNA-

E. coli= ~ 11% (average intergenic distance = 130 bp)

Human = > 95% (there are islands, hundreds of thousands of bp, apparently without a gene.)

Eukaryotic Genomes:

• Contain massive amounts of repetitive DNA sequences (Define).

• Human- repeat seqeunces comprise over 50% of genome.

• E. coli- DNA is almost entirely unique

What are the human repetitive DNA sequences?

1) Simple ‘stutters’ (CAGCAGCAGCAGCAGCAG . . . .)

2) Psuedogenes

3) Transposable elements (= > 40% of HG)

4) Segmental duplications (~ 10 - -300 kb)

5) Gene Families (maybe a reflection of genomic duplications)

Shocking discovery in mid 1970s:

Eukaryotic genes are interrupted by noncoding DNA!

Almost all transcripts (mRNA) are spliced before leaving the nucleus.

Exon

=

Genetic code

Intron

=

Non-essential DNA ? ?

• The mechanism of splicing is not well understood.

Variable mutation rate?

• Most mutations in introns and intergenic DNA are (apparently) harmless

• Consequently, intron and intergenic DNA sequences diverge much quicker than exons.

Shocking discovery in late1990s:

• Some eukaryotic genomes have thousands of genes that are alternatively spliced.

• In the human genome, it is now estimated that 35% of the genes undergo alternative splicing

Alternate Splice sites generate various proteins isoforms

Bacteria cells are different:

• Prokaryotic cells- No splicing (i.e. – no split genes)

• Eukaryotic cells- Intronless genes are rare (avg. # of introns in HG is 3-7, highest # is 234); dystrophin gene is > 2.4 Mb.

Identifying all of the human genes

a) Is tough

b) Is easy

c) Is really tough

Making it tough:

• Pseudogenes

• Large intergenic regions

• Prevelant and long introns

• Alternative splicing

Comparison of 4 plant genomes:

8 genes in C. elegans- 5 intronic genes:

12 of the 64 genes duplicated between human chr. #18 and #20

Is there a gene in there? 5’

CAGACTGTAGTCGTAGTCGTGTAGTCGTATGGCCGTAGTCGTAGTCGATCGTGATTCGTAGTCGTAGTCGTAGTCGTAGTCGTAGTCGTAGTCGAGTCGTAGTCGTAGTCGTAGTCGTAGCTGTAGTCGTAGTCGTAGTCGAGTCGTAGTCGTGTACGTGTAGTCGTAGTCGTAGCTGTACTAGTCGTATGCGTAGTCGTAGTCGTAGCGAGTCTGAGTGTACGTCGTAGTGCTAGTTGCGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGCTGTAGTCGTAGTCGTAGTCGTAGTCGTGTACGTAGTGTCGTATGCGAGGCTAGTCAGGTCGTATGGCTAGTATGCGTAGTCGAGTCGTAGTCGTAGTGTACGTCGTAGTGTCAGTCGTCAGTTGACGTACGTAGTGTCGTAGTCGTAGTCGTAGTCGTAGTCGTAGTGAGTGTACGTTGCGTATGGCTATGTATGTGCAGTGCTGTAGTCGTAGTGCTGTAGTCAGTTGCGTAGTGATGTACGTGTATGCGTATGCGTAGTCTGAGTTGCTGAGTGCTAGTCTGAGTGTCGTAGTCGTAGTGCGTAGTCGTATGCGTATGCGTATCGGATTGCGTAGTGTAGCTGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGTCAGTCGTGTAGTAGTCGTATGACCGCGGCGCGAGTTGGTGCGGCGGGGGCTATTTTTCGGAGCGTGTAAGGTTATTAGGTTTTTCCTATTATATGCGCTTAGCGTAGCGCGATTAGCGTATAGCGCATTATATATGCGCCTTCTCTCTTCGAGAGATCTCAGCGTCGTAGTGTACGTCGT

CGAGGCACTGTAGTCGTAGTCGTGTAGTCGTATGGCCGTAGTCGTAGTCGATCGTGATTCGTAGTGGTAGTCGTAGTCGTAGTCGTAGTCGTAGTCGAGTCGTAGTCGTAGTCGTAGTCGTAGCTGTAGTCGTAGTCGTAGTCGAGTCGTAGTCGTGTACGTGTAGTCGTAGTCGTAGCTGTACTAGTCGTATGCGTAGTCGTAGTCGTAGCGAGTCTGAGTGTACGTCGTAGTGCTAGTTGCGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGCTGTAGTCGTAGTCGTAGTCGTAGTCGTGTACGTAGTGTCGTATGCGAGGCTAGTCAGGTCGTATGGCTAGTATGCGTAGTCGAGTCGTAGTCGTAGTGTACGTCGTAGTGTCAGTCGTCAGTTGACGTACGTAGTGTCGTAGTCGTAGTCGTAGTCGTAGTCGTAGTGAGTGTACGTTGCGTATGGCTATGTATGTGCAGTGCTGTAGTCGTAGTGCTGTAGTCAGTTGCGTAGTGATGTACGTGTATGCGTATGCGTAGTCTGAGTTGCTGAGTGCTAGTCTGAGTGTCGTAGTCGTAGTGCGTAGTCGTATGCGTATGCGTATCGGATTGCGTAGTGTAGCTGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGTCAGTCGTGTAGTAGTCGTATGACCGCGGCGCGAGTTGGTGCGGCGGGGGCTATTTTTCGGAGCGTGTAAGGTTATTAGGTTTTTCCTATTATATGCGCTTAGCGTAGCGCGATTAGCGTATAGCGCATTATATATGCGCCTTCTCTCTTCGAGAGATCTCAGCGTCGTAGTGTACGT

CAGACTGTAGTCGTAGTCGTGTAGTCGTATGGCCGTAGTCGTAGTCGATCGTGATTCGTAGTCGTAGTCGTAGTCGTAGTCGGGCTTGTAGTCGAGTCGTAGTCGTAGTCGTAGTCGTAGCTGTAGTCGTAGTCGTAGTCGAGTCGTAGTCGTGTACGTGTAGTCGTAGTCGTAGCTGTACTAGTCGTATGCGTAGTCGTAGTCGTAGCGAGTCTGAGTGTACGTCGTAGTGCTAGTTGCGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGCTGTAGTCGTAGTCGTAGTCGTAGTCGTGTACGTAGTGTCGTATGCGAGGCTAGTCAGGTCGTATGGCTAGTATGCGTAGTCGAGTCGTAGTCGTAGTGTACGTCGTAGTGTCAGTCGTCAGTTGACGTACGTAGTGTCGTAGTCGTAGTCGTAGTCGTAGTCGTAGTGAGTGTACGTTGCGTATGGCTATGTATGTGCAGTGCTGTAGTCGTAGTGCTGTAGTCAGTTGCGTAGTGATGTACGTGTATGCGTATGCGTAGTCTGAGTTGCTGAGTGCTAGTCTGAGTGTCGTAGTCGTAGTGCGTAGTCGTATGCGTATGCGTATCGGATTGCGTAGTGTAGCTGTAGTCGTAGTCGTAGTGTCGTAGTCGTGTAGTCAGTCGTGTAGTAGTCGTATGACCGCGGCGCGAGTTGGTGCGGCGGGGGCTATTTTTCGGAGCGTGTAAGGTTATTAGGTTTTTCCTATTATATGCGCTTAGCGTAGCGCGATTAGCGTATAGCGCATTATATATGCGCCTTCTCTCTTCGAGAGATCTCAGCGTCGTAGTGTACGTCGC

3’

How to confirm the identification of a gene?

• Possible answer- Identify the gene by identifying its promoter.

Promoters are DNA regions that control when genes are activated.

Promoter coding region

[ ]

• Exons encode the information that determines what product will be produced.

Promoters encode the information that determines when the protein will be produced.

Nucleotides of a particular gene are often numbered:

Demonstration of a consensus sequence.

• De

Three current bioinformatic challenges:

• 1) verification of the data (it is correct?)

• 2) Thorough annotation of the data (includes developing appropriate means of annotating)

• 3) How to handle data of ever-larger chunks

A dot = a promoter. Dark purple = left to right, light purple = right to left. Overlapping

genes= green

•

Inner circle = ccw direction, outer circle = cw direction

How to find a gene?

• Look for a substantial ORFs and associated ‘features’.

ORFs- open reading frames

• Two nucleic acids, that are exact complements of each other will hybridize.

• Two nucleic acids that are mostly complementary (some mismatchs) will . . .

. . . hybridize under the right conditions.

Recombinant DNA techniques?

• Many popular tools of recDNA rely on the principle of DNA hybridization.

• In large mixes of DNA molecules, complementary sequences will pair.

Hybridization ‘in silico’

• Algorithms have been written that will compare two nucleic acid sequences. Two similar DNA sequences (they would hybridize in solution) are said ‘to match’ when software determines that they are of significant similarity.

8/10= 80%

Mouse ATGCCGTGCTA

: : : : : : : :

Human ATG--CGGGCAA

Protein- Protein similarity searches?

• Many algorithms have been designed to compare strings of amino acids (single letter amino acid code) and find those of a defined degree of similarity.

60 70 80 90

#1 TSIDQLRATTSYDELRQDGSTTISYDDYSR : : : . : : : : : : : : : : : : : : : : : : : : .

: : : : :#2 TSIEQLRATTSYDELRQDGSTTISTDDYSR

Significance of sequence similarity

• DNA similarity suggests:

• Similar function

• Similar structure

• Evolutionary relationship

The End