Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.     

Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5'' exons and possible mechanism for the genesis of the 3'' end of v-src.

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.     

Studies on the organization of the human apolipoprotein B 100 gene

The organization of five exons of the 3' terminal end of the human apolipoprotein B 100 (apo B 100) gene 1906, 184, 115, 7572 and 374 bp long have been determined from two overlapping EMBL3 human genomic clones extending over 18 kb. They encode more than 70% of the apo B 100 amino-acid sequence. The introns between these five exons were sequenced revealing the common intron/exon splice junction sequences. The 7572 bp exon is the longest exon so far reported for mammalian genes with the proposed sequence coding for the LDL receptor binding site. Its possible relationship to apolipoprotein B 48 is discussed.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Studies on the structures of the normal and abnormal goat thyroglobulin genes

We have cloned the thyroglobulin (Tg) gene of normal goats and goitrous goats which have a Tg synthesis defect. At the 5'-end of the gene, we studied cosmid clones covering a region from 20 kilobases (kb) upstream from the Tg gene to 42 kb into it. Electron microscopy and restriction mapping show that this part of the gene contains 20 exons of 90-1190 bp, in total 4.9 kb of exonic information (56% of the mRNA) split by 19 introns of 150-9100 bp. The exons comprise 12% of the 5' sequences cloned. At the 3'-end, 55 kb were cloned, containing 10 kb of the gene which comprises only 3 exons of 550 bp in total. Sequence analysis of the 3'-end of the normal and abnormal Tg genes has revealed one transition mutation 3' to the reading frame in a stem-loop structure region of the last exon near the poly(A) addition site. Analysis of the promoter site and the first 5 exons has revealed only one difference between the normal and goitrous Tg genes: a Ser----Leu transition in exon 5. We also found an insertion in the fifth intron of the abnormal gene.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trans splicing of mRNA precursors in vitro

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.     

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.     

Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.