RESUMO
Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.
Assuntos
Anexinas/genética , Regiões Promotoras Genéticas , Motivos de Aminoácidos , Sequência de Aminoácidos , Anexinas/química , Anexinas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetidas Terminais , Transcrição Gênica , TransfecçãoRESUMO
The genomic organization of the annexin A11 gene was determined in mouse and human to assess its congruity with other family members and to examine the species variation in alternative splicing patterns. Mouse annexin A11 genomic clones were characterized by restriction analysis, Southern blotting, and DNA sequencing, and the homologous human gene (HGMW-approved gene symbol ANXA11) was deciphered from high-throughput genomic sequence with coanalysis of expressed sequence tags. Exons 6-15 of the tetrad core repeat region differ from annexins A7 and A13 but are spliced identically to other phylogenetic descendents, making annexin A11 the putative primary progenitor of up to nine paralogous human annexins. The 5' regions consist of untranslated exon 1, followed by an extensive intron 1 comprising almost half the total gene length of >40 kb, and additional GC-rich exons 2-5 encoding the proline- and glycine-rich amino-terminus. Distinct cDNA isoforms in cow and human were determined to be unique to each species and hence of dubious general significance for this gene's function. Multiple transcription start sites were revealed by primer extension analysis of the mouse gene, and transfection constructs containing the prospective promoter generated transcriptional activity comparable to that of the SV40 promoter. Internal repetitive elements and vicinal gene markers were mapped for the complete human annexin A11 gene sequence to characterize the surrounding genomic environment.
Assuntos
Anexinas/genética , DNA Complementar/genética , Genes/genética , Células 3T3 , Processamento Alternativo , Animais , Sequência de Bases , DNA/química , DNA/genética , DNA/isolamento & purificação , DNA Complementar/química , Éxons , Ligação Genética , Humanos , Íntrons , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Mouse annexin V genomic clones were characterized by restriction analysis, Southern blotting and DNA sequencing. The entire gene spans close to 50 kb of the mouse genome and contains 14 exons ranging in size from 31 bp for exon 2 to 482 bp for exon 13 up to the polyadenylation site. Intron sizes range from 111 bp for intron 1b to more than 17 kb for intron 2. Non-coding exon 1 is present in two alternative forms separated by approx. 7.4 kb, and the two promoters associated with exons 1a and 1b are quite distinct. The upstream promoter has a TATA box and may direct the limited, tissue-specific expression of mRNA transcripts containing exon 1a. The downstream, TATA-less promoter has high G+C content, and exon 1b predominates among abundantly expressed mRNA species. The conservation of certain cis-elements, including Sp1, AP2, gamma-IRE and NF-IL6, in orthologous species of annexin V genes points to their possible role in trans-acting protein factor binding and gene regulation. Primer-extension analysis revealed multiple origins for transcription, with principal start sites 100-150 bp upstream of the ATG start codon in exon 2. Intron 4 was longer than that previously identified in the orthologous rat gene due to the integration of an apparently complete copy of the murine endogenous retrovirus element, MuERV-L. Phylogenetic analysis of annexin V from 12 species and the presence of neighbouring loci with paralogous counterparts linked to annexin VI pointed to the common ancestry of these genes via chromosomal duplication more than 600 million years ago.