Part:BBa_K3952001

APOB-100 (Cholesterol Deficiency Allele)

Located within chromosome 11 of the Bos taurus genome, the APOB gene codes for Apolipoprotein B, a protein involved in the transport of cholesterol in the bloodstream. Studies have reported an inheritable allele within the APOB gene that causes cholesterol deficiency (CD) in cattle. This allele is created by the insertion of a transposon, ERV2-1 between the 24th and 25th nucleotide of exon 5, The insertion polymorphism results in cholesterol deficiency (CD) within cattle.

This part contains a fragment of the cholesterol deficiency allele of the APOB gene with an ERV2-1 transposon insertion between nucleotides CT at positions 221 and 222. The wildtype version of the APOB gene (part number BBa_K3952000) contains no insertion between the nucleotides CT at positions 221 and 222.

The APOB gene (Gene ID 494004) was located within the genomic DNA sequence of the Bos taurus, using the NCBI reference assembly.

Figure 1: The CD APOB positive control DNA. The nucleotides highlighted in yellow indicate the location of the 24th and 25th nucleotides in exon 5. The ERV2-1 transposon is indicated in blue text, and the altered illegal SpeI restriction site is shown in bold blue. The wildtype APOB positive control is the same sequence, minus the insert. The wildtype and the CD APOB positive control constructs have been entered into the iGEM parts registry as BBa_K3952000 and BBa_K3952001.

Usage and Biology

The purpose of this part is to serve as a positive control for a Flappase genetic testing system. The system requires the amplification of DNA around the polymorphism, therefore the sequence provide is only a fragment of the Bos taurus genome. The entire sequence of the gene was not cloned to only showcase where the allele would be present (exon 5), and to facilitate ease of cloning.

This part does contain the APOB inheritable allele. The allele is created by the insertion of a transposable element (ERV2-1) into the APOB gene. The insertion occurs between the 24th and 25th nucleotide of exon 5, which is between nucleotides CT at positions 221, 222. The insertion polymorphism results in cholesterol deficiency (CD) within cattle.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 330
  Illegal XhoI site found at 591
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 853