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Acetyl CoA carboxylase splice variant and uses thereof

A variant, compound technology, applied in the field of isolated nucleic acid molecules, which can solve problems such as unclear mechanism

Inactive Publication Date: 2007-06-13
ASTRAZENECA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism is unclear, as the attachment of ACC2 monomers / protomers to the mitochondrial outer membrane is expected to limit how the enzyme polymerizes

Method used

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  • Acetyl CoA carboxylase splice variant and uses thereof
  • Acetyl CoA carboxylase splice variant and uses thereof
  • Acetyl CoA carboxylase splice variant and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0110] In silico (insilico) prediction of a novel splice variant ACC2(1b) of human acetyl-CoA carboxylase 2

[0111] Using a combination of new sequence information and in silico prediction methods, the sequence of a new splice variant of human ACC2 was identified. For EMBL entries containing human genomic DNA, blast the human ACC2 EMBL entry HSU89344 (Blast2 NCBI). The human BAC clone RCPI11-443D10 from chromosome 12 was found to contain the gene encoding human ACC2. The genome sequence of this clone was found in EMBL entry AC007637.

[0112] Using the alignment between HSU89344 and AC007637, the gene structure of human ACC2 was analyzed. Using alignments, exons are predicted, and using intron splice consensus sites, accurate exon boundaries are predicted. Alignment shows that the human ACC2 gene consists of 52 coding exons. Between the sequence of HSU89344 and the genome sequence of AC007637, multiple mismatches were found. The corrected sequence of human ACC2 was gener...

Embodiment 2

[0122] Cloning of human ACC2

[0123] To develop a method for the production of recombinant ACC2, cDNA encoding human ACC2 was cloned by RT-PCR from human skeletal muscle mRNA. A total of 3 fragments covering the 7.5 kb sequence of ACC2 were PCR amplified and multiple clones were sequenced.

[0124] The first fragments 1 to 3925 were PCR amplified from a human cardiac cDNA library using the proofreading polymerase pfu (Stratagene). The primers used in PCR were 5'-ATGGTCTTGCTTCTTTGTCTATC-3' (SEQ ID NO: 6) as a forward primer and 5'-GGCTGTTTAACACATAGGCGA-3' (SEQ ID NO: 7) as a reverse primer. The product was cloned into "TA" cloning vector pCR2.1, transformed into Escherichia coli (E.coli) XL-10 Gold cells (Stratagene), and subjected to complete double-strand sequencing.

[0125] The second and third fragments, 3250 to 5356 bp and 5335 to 7302 bp, were PCR amplified from human skeletal muscle cDNA using Taq-plus Precision (Stratagene), respectively. For the second PCR fragmen...

Embodiment 3

[0131] TaqMan analysis of human tissue confirms the presence of ACC2(1b) transcripts

[0132] Oligonucleotide primers and probes for hACC2(1b) were designed using Primer Express 1.5 software (Applied Biosystems). Design of forward primer 5'-AGTCCTGCCAAGTGCAAGATCT-3' (SEQ ID NO: 12), reverse primer 5'-TCTGTGCAGGTCCAGCTTCTT-3' (SEQ ID NO: 13) and FAM-labeled probe 5'-TGATCGCGAAGTAAAGCCGAGCATGT-3' (SEQ ID NO: 14) to cover exon 1B and exon 2. Human acid ribosomal phosphoprotein (h36B4) primer and VIC-labeled probe were used as endogenous controls.

[0133] First-strand cDNA was synthesized using SuperscriptIII (Invitrogen) and oligoDt primers from 100 ng polyA+RNA (adipose tissue) and 1 μg total RNA (cardiac muscle, skeletal muscle and liver). To determine the expression of ACC2(1b) in human cardiac muscle (Ambion), skeletal muscle (Ambion), liver (BD Biosciences) and adipose tissue (BD Biosciences), real-time PCR (ABI prism 7500 detection system, Applied Biosystems) was used. ...

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Abstract

The present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an acetyl CoA carboxylase 2 (ACC2) splice variant. It also relates the corresponding polypeptide encoded by said nucleic acid and to methods of identifying a compound potentially useful for treating diseases or disorders associated with impaired ability to oxidise fatty acids, which comprises assaying the compound for its ability to modulate the activity or amount of a ACC2 splice variant complex or a complex thereof.

Description

technical field [0001] The present invention relates to isolated nucleic acid molecules comprising a nucleotide sequence encoding an acetyl-CoA carboxylase 2 (ACC2) splice variant. Also contemplated are corresponding polypeptides encoded by said nucleic acids, and methods of identifying compounds potentially useful in the treatment of diseases or disorders associated with impaired ability to oxidize fatty acids, comprising determining that the compound modulates the ACC2 splice variant complex or its The activity or quantitative capacity of the complex. Background of the invention [0002] Many recent discoveries have pointed to alternative pre-messenger RNA splicing as generators of protein diversity, which in some cases rivals the immune system in the degree of diversity generated by single genes. Based on genome-wide analysis of alternative splicing, as many as 40-60% of human genes have been predicted to have alternatively spliced ​​forms. This process allows for the o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C07K16/40G01N33/573A61P3/04A61P3/10C12Q1/68
CPCC12Q2600/156G01N2800/042C12N9/93G01N2333/9015C12Y604/01002G01N2800/044G01N2500/00C07K16/40A61P3/04A61P3/06A61P3/10A61P43/00
Inventor J·克拉法姆B·科尼利厄森S·哈伦
Owner ASTRAZENECA AB
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