Chapter 6 Genomics and Gene Recognition

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Chapter 6 Genomics and Gene Recognition
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• The sequencing of prokaryotic genomes can provide
  insights into the minimum requirements of life.
• Most of the prokaryotic genome is devoted to
  maintenance of a cells basic infrastructure.
• The smallest bacteria to be sequenced is
  Haemophilus influenzae

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• Haemophilus influenzae has about 256 to 300
  genes.
• The DNA sequencing technology that made it
  possible to determine the number of genes in
  Haemophilus influenzae is based on the contigs
• Consider the E.coli genome which is 4.6 million
  nts in length.

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• Current DNA sequencing technology produces a
  continuous DNA of 1000 nts.
• So to sequence a genome that is 4.6 million nts
  would ideally require 4,600 sequencing reactions.
• When the E. coli genome is cloned it is not
  cloned as one contiguous piece of DNA but many
  different pieces of DNA which overlap.

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• It is necessary to arrange these DNA sequences
  into a contiguous DNA sequence, i.e. contig.
• The question arises how many DNA sequence
  reactions are required to assemble contigs for a
  genome?

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• The chance of sequencing anywhere in a 4.6
  million genome is 1000/4,600,000 X 100
• While the chance of missing a region of the 4.6
  million genome is 4,599,000/4,600,00 x 100
• Therefore the number of clones from a genomic
  library that need to sequenced to cover 95 of
  the genome.

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• (4,599,000/4,600,000)N 0.05
• So we need to sequence 20 million additional nts
  to have a 95 chance of sequencing the bacterial
  genome.

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• The structure of a typical prokaryote gene.

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• The prokaryotic RNA polymerase consists of
  several different proteins.
• Beta prime ( ) a protein involved in
  binding to DNA template
• Beta ( ) to link one nt to another
• Alpha ( ) to hold all subunits together
• Omega ( ) recognizes the promoter sequence

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• The sigma protein varies significantly between
  bacteria.
• It is directly responsible for a cells ability
  to turn off sets of genes.
• By examining the promoter sequences we can
  determine which genes will be turned off by a
  particular sigma protein.

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• Sigma 70 General TTGACA
• Sigma 32 Heat Shock CTTGAAA
• Sigma 54 Nitrogen stress CTGGCAC
• These sequences are part of the -35 consensus
  sequence in bacteria.
• The -35 and -10 sequences are recognized by sigma
  protein and comprise the consensus sequence.

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• Computer programs can search for different census
  sequences
• Comparing different Promoter sequences can be
  used to rank different operons in terms of
  expression.

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• There 64 possible codons out of which three UAA,
  UAG and UGA are stop codons.
• Stop codons occur in a non-coding DNA sequence
  about every 21 codons.
• So a sequence of DNA that is at least 30 codons
  in length without a stop codon is a open reading
  frame.

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• If codons occur randomly in a DNA sequence. The
  chance of a DNA sequence not containing a stop
  codon is
• (61/64)N where N is the number of codons.
• A 95 confidence that a stretch of codons does
  not contain a stop codon is
• (61/64)N 0.05 or 60