Homologous recombination between the LTRs of a human retrovirus-like element causes a 5-kb deletion in two siblings.

The RTVL-H family of human endogenous retrovirus-like elements consists of approximately 1,000 intact members and of a similar number of solitary long terminal repeats (LTRs). In this study, the genetic heterogeneity of these elements has been investigated using unique flanking probes isolated from cDNA clones containing RTVL-H sequences. Four such probes were used to screen a panel of human DNA samples for genetic differences. One of these probes detected a 5.0-kb deletion in two related individuals. Cloning and DNA hybridization analysis indicated that the nondeleted common allele contained an intact RTVL-H element, whereas the deleted variant allele contained only a single LTR. DNA sequence comparisons strongly suggest that the deletion is due to homologous recombination between the 5' and 3' LTRs of the RTVL-H sequence. This is the first reported case of a DNA variation in humans that is due to an LTR-LTR excision event.     

Isolation and structural mapping of a human c-src gene homologous to the transforming gene (v-src) of Rous sarcoma virus.

We have utilized a lambda Charon 4A human genomic library to isolate recombinant clones harboring a highly conserved c-src locus containing nucleotide sequences homologous to the transforming gene of Rous sarcoma virus (v-src). Four overlapping clones spanning 24 kilobases of cellular DNA were analyzed by restriction endonuclease mapping. Human c-src sequences homologous to the entire v-src region are present in a 20-kilobase region that contains 11 exons as determined by restriction mapping studies utilizing hybridization to labeled DNA probes representing various subregions of the v-src gene and by preliminary DNA sequencing analyses. A considerable degree of similarity exists between the organization of the human c-src gene and that of the corresponding chicken c-src gene with respect to exon size and number. However, the human c-src locus is larger than the corresponding chicken c-src locus, because many human c-src introns are larger than those of chicken c-src. alu family repetitive sequences are present within several human c-src introns. This locus represents a highly conserved human c-src locus that is detectable in human cellular DNAs from various sources including placenta, HeLa cells, and WI-38 cells.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Molecular cloning, characterization, and expression of a human 14-kDa lectin

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.     

Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.