Characterization of Five Novel Genes Expressed in the Heart

Autor: Lian-ju Lin, 林蓮汝
Rok vydání: 2002
Druh dokumentu: 學位論文 ; thesis
Popis: 90
In an effort to isolate genes which may play a role at different stages of the human heart, two libraries were constructed using subtractive hybridization between normal fetal (gestational age 13wk-28wk) and adult (27 years of age) hearts. In this thesis work, cloning and characterization of five clones, a95, a202, c11, c306, and c324, from these two libraries are described. The prefix〝c〞represents genes expressing at a higher level during adult stage, while〝a〞denotes the opposite. Each of these clones was extended by 3' and 5' RACE (rapid amplification of cDNA ends) to give the full-length coding sequence. Tissue expression pattern was investigated by real-time quantitative polymerase chain reaction using cDNAs reverse transcribed from mRNAs of various adult and fetal tissues. Their genomic organization and chromosome assignment were determined by comparing full-length cDNA sequence with HTGS (high-throughput genomic sequence) database. In addition, bioinformatic tools were employed to predict their possible biological functions based on amino acids sequence. The c306 cDNA is 5484 bp, consists of 41 exons and 40 introns at human chromosome 10q25.3, and codes for a deduced peptide of 132.3 kDa with a pI of 9.12. Sequence analysis using BLAST (basic local alignment search tool) search, c306 shares 86.65% similarity with the mouse nebulin-related anchoring protein (N-rap). They both contain one LIM domain and twenty-six NEBU repeats and are expressed in the heart and skeletal muscle. These results suggest that c306 should be the human N-RAP. NEBU repeats are known to bind to actin, and the LIM domain can interact with another protein. Therefore, it is suggested that N-rap may serve as a connection between actin filaments and proteins at sarcolemma. Examination of 1.5 kb upstream genomic sequence revealed potential binding sites for a variety of transcription factors such as CSX, MyoD, AP4, C/EBP and GATA。 The 2527-bp c11 cDNA consists of 6 exons and 5 introns at human chromosome 10q25.3, and contains an intact open reading frame that encodes 444 amino acids with a predicted molecular weight of 49.1 kDa. The presence of a CpG island at its immediate 5’ suggests that the current 5’ end is very close to the bona fide transcription start site. Sequence comparison through BLAST search, c11 is identical to a human mRNA clone, NM_024942. Expression level is higher in the adult heart than that in the fetal heart. In addition, brain, colon, testis of adult and brain of fetal have abundant expression. Examination of 1.5 kb upstream genomic sequence revealed potential binding sites for a variety of transcription factors such as CSX、 AP1、AP4、GATA and C/EBP. The c324 cDNA, which is 2195 bp in length, predicts a protein of 585 amino acids sharing 99.82% similarity to CDA02. It consists of 14 exons and 13 introns at human chromosome 3q24. The presence of a CpG island immediate at its 5’ suggests that the current 5’ end is very close to the real transcription start site. In the aspect of tissue expression pattern, expression level in the adult heart is higher than that in the fetal heart. In addition, brain, kidney and skeletal muscle have abundant expression. Examination of 1.5 kb upstream genomic sequence revealed potential binding sites for a variety of transcription factors such as CSX, MyoD, c-Myb, GATA, AP1, C/EBP and CRE-BP. There are two splicing forms with a size of 2544 bp and 2864 bp, resprctively, for a95. Both consist of at least 12 exons and 11 introns and encode 431 amino acids. It is mapped to human chromosome 12q24.11. Sequence comparison through BLAST search revealed a 94.44% similiarity with a mouse protein, CDV1-R. In terms of tissue expression pattern, expression level in the adult heart is similar to that in the fetal heart. In addition, skeletal muscle and testis have abundant expression. Examination of 1.5 kb upstream genomic sequence revealed potential binding sites for a variety of transcription factors such as CREB、E1K-1、c-Myc、c-Myb、E2F、SRY、GATA、AP-1、C/EBP and SP1. The transcript of a202 is 3192 bp in length. The genomic organization and chromosome assignment of a202 determined by comparing full-length cDNA sequence with HTGS showed that a202 contained 18 exons and 17 introns at human chromosome 3p21.1. The presence of a CpG island immediate at its 5’ suggests that the current 5’ end may be very close to the exact location of transcription start site. Sequence comparison through BLAST search, a202 is similar to a human mRNA clone, LOC131827. In terms of tissue expression pattern, expression level in the adult heart is higher than that in the fetal heart. In addition, adult brain, liver, pancreas, placenta, skeletal muscle, ovary and testis also express the transcript abundantly. The expression level in the fetal tissues is very low. Examination of 1.5 kb upstream genomic sequence revealed potential binding sites for a variety of transcription factors, including CSX、MyoD、AP-1、C/EBP and GATA.
Databáze: Networked Digital Library of Theses & Dissertations
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