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.     

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.     

Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.