Abstract
□摘要：基膜素乃基底膜中之一主要組成物；它是一個大分子醣蛋白，由其次級單位結構甲，乙一及乙二等三條鏈互以雙硫鍵相聯而成。基膜素在細胞的發育過程，如細胞的黏合，遷移，分裂，生長，和分化等現象上扮演了重要的角色。本實驗室分離出一長5737核□的果蠅基膜素乙二鏈cDNA殖株，並將其定序完畢。推演其胺基酸序列顯示成熟而未醣化的胜□長度為1606個胺基酸(Mr=178,665)。此乙二鏈含有100 個cysteine，大部份集中於兩個cysteine- 豐富區；以及11個N-X-S 或N-X-T 序列，可能是氮基聯結之醣化作用處。有一Y-F-G-S-R 序列，與人類，老鼠及果蠅乙一鏈中之細胞－結合胺基酸序列非常類似，亦出現在果蠅乙二鏈的相對位置。推演此鏈的二級結構顯示有六個不同的結構區，其中有兩個（圖表省略）－螺旋區，兩個具相似重複段落的cysteine- 豐富區，以及兩個球狀區。果蠅的乙二鏈與人類及老鼠的乙二鏈比較，分別有40.3及41.1％的胺基酸相同；若與人類，老鼠及果蠅的乙一鏈比較，則相似程度分別為29.6，30.0，及29.4％。果蠅基膜素乙二鏈基因包括一長10.6kb的5端空白區；一長9kb的結構基因，共有十個表現序列；及一長7.4kb 的３端空白區。內在序列的大小自30bp至2.08kb，變化很大。第一表現序列只解讀出54bp的５端不轉譯區，隨後緊接著就是最大的插入序列。轉譯起始密碼ATG 及停止密碼TAG 分別落在第二和第十表現序列。其2.1-kb起動子區在-35 處並沒有TATA盒，但是在-100處有一CAAT盒。然而在-640處有一TATA盒，另外在-660處有另一CAAT盒。有二個5'-TAACC-3' 序列，與老鼠基膜素乙一鏈中視黃酸對應子(RARE)的主要結合序列類似，亦出現在果蠅乙二鏈的相對位置。在-1310 及-1450核□區，有兩個序列5'-CGTC(T/G)C(G/R)-3' ，若將之反互補，則類似cAMP對應子(CRE) 序列。另外一些有趣的特徵是，在-30 至-70 核□區，有一個與果蠅fos-或jun-相關抗原(dFRA,dJRR) 之結合序列類似的序列，及兩個與zeste-結合序列相似的序列。此外，在-100區的CAAT盒之前，及在zeste-結合序列之前，且在dFRA和dJRA結合序列之前，各有兩段具６個核□的重復片段。最後，在此起動子區有五個5'-GAGAG-3' 序列，與2/3 的GAGA因子結合序列類似。此2.1-kb起動子片段接入一氯黴素乙醯轉移□基因的前端，作成重組質體DNA後轉殖入Schneider line ll (SL-2)細胞。這些細胞經視黃酸或╱和cAMP刺激之後，其活性較之未受藥物刺激者高3-3.5 位。此結果顯示，此一特殊的起動子所含的DNA序列，可能對發育過程的調節有所幫助。///////ABSTRACTLaminin, a major component of basement membrane, is a large glycoproteinconsisting of three disulfide-bonded subunits, A,B1, and B2.Laminin is involved in a number of biological activities includingpromotion of cell adhesion, migration, mitogenesis, growth, anddifferentiation. We have isolated and sequenced a Drosophila laminin B2chain cDNA clone that spans 5737 nucleotides. The deduced amino acidsequence predicts that the mature and nonglycosylated polypeptide has achain length of 1606 residues (Mr=178,665). This B2 chain contains 100halfcystine residues, most of which are located in two cysteine-richdomains, and 11 N-X-S or N-X-T sequences which are potential sites ofN-linked glycosylation. A Y-F-G-S-R sequence, which is similar to thecell-binding amino acid sequences in the human, mouse, and Drosophila B1chains, is present at a comparable site in the Drosophila B2 chain. Thepredicted secondary structure reveals the presence of six structurallydistinct domains, of which two are mainly（圖表省略）two are cysteine-rich with homologous repeats, and two are globularregions. The Drosophila B2 chain is 40.3 and 41.1% identical to the humanand mouse B2 chains, respectively, and 29.6, 30.0, and 29.4% identical tothe Drosophila, human, and mouse B1 chains, respectively.The structures of the Drosophila laminin B2 chain gene was determined from8 overlapping genomic clones that spanned 27 kilobase pairs (kb),including 10.6 kb of the 5'-flanking region, 9 kb of the structural gene,and 7.4 kb of the 3'-flanking region. The transcription initiation sitewas determined by primer extension and S1 nuclease mapping. DNA sequencingdemons-trated that the gene consists of 10 exons. The intron sizes varyfrom 30 base pairs (bp) to 2.08 kb. The first exon encodes only 54bp of the 5'-untranslated region, then comes the first and largest intron.The translation start condon ATG and stop codon TAG are located at thesecond and the tenth exons, respectively. The 2.1-kb promoter region doesnot contain TATA box at-35, but it has a CAAT box-like sequence at-100.However, a TATA box-like sequence is present at-640, and another CAATbox-like sequence at-660. Two 5'-TAACC-3' sequences, that are similar tothe consensus motifs present in the binding region for the retinoic acidresponse element of the mouse laminin B1 gene, are present in theDrosophila counterparts. Two reverse complementary cAMP response elementlike sequences, 5'-CGTC(T/G)C(G/A)-3'can be found at-1310 and -1450. ADrosophila fos-or jun-related antigen (dFRA, dJRA) binding sequence andtwo zeste binding sequences are present at -30 to -70. Furthermore,thereare two direct repeats of 6 nucleotides present in front of the -100 CAATbox, and one of the zeste binding, and also the dFRA and dJRA bindingsequences. Additionally, 5'5-GAGAG-3' sequences, which are similar to 2/3of the GAGA factor binding sequence, are present in the promoter region.A recombinant plasmid containing the 2.1-db promoter segment coupled tothe chloramphenicol acetyl transferase (CAT) gene was constructed andtransfected into Schneider line II (SL-2) cells. The SL-2 cellstransf-ected with this construct induced by retinoic acid and/or8-Bromo-cAMP showed 3-3.5 folds increase of CAT activity when compared tothe noninduced cells. These results indicate that this structurally uniquepromoter contains DNA sequences that may help regulate this gene duringdevelopment.