Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.     

Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase

Complementary DNA clones for human glucocerebrosidase were isolated from a human hepatoma library in lambda gt11. The complete nucleotide sequence of the 1805-base pair cDNA insert has been determined. In addition to 5' and 3' untranslated regions (51 and 206 base pairs, respectively), the cDNA insert contains 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylation sites are identified. Examination of the 19 amino acids of the leader polypeptide beginning with the ATG at position 52 revealed a hydrophobic core and a carboxyl-terminal glycine at the peptidase cleavage site, features consistent with the leader sequences described for other human translocated proteins. The Mr of 57,000 calculated from the 516 amino acids deduced from cDNA sequence is in good agreement with that identified by immunoprecipitation following in vitro translation of human placental mRNA.     

The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.     

Human tissue factor: cDNA sequence and chromosome localization of the gene

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultraviolet light-induced cleavage of DNA in the presence of iodoHoechst 33258: the sequence specificity of the reaction.

IodoHoechst 33258 sensitizes DNA to cleavage by near ultraviolet light (UV-A). Following an earlier study which showed that the UV-induced cleavage is at discrete locations corresponding to the ligand binding sites, this paper reports a more extensive analysis of the sequence specificity of cleavage. The experiments involved use of double-stranded DNA synthesised on primed M13 templates. Analysis of cleavage in a 280bp sequence in M13mp18 and a 310bp sequence in three M13mp9 clones ('alpha-32', 'alpha-82' and 'alpha-22') derived from human alpha-DNA, showed that for all of the twenty-nine strong and very strong damage sites, cleavage was at the 3' end of a run of three or more consecutive AT base pairs. The extent of cleavage was higher for sites with consecutive Ts than for consecutive As, and greatest for the sequence cTTTTca. Comparison of three closely-related alpha-DNA clones enabled assessment of single bp changes and essentially confirmed the results of detailed analysis of binding cleavage sites in mp18 and alpha-32. Decreasing the input ratio of iodoHoechst/per bp DNA from 0.13 to 0.013 altered the sequence specificity, and sites possessing only three consecutive AT bps were generally not cleaved. The contributions of both the strength of ligand binding and the efficiency of photolytic cleavage to the overall extent of cleavage by UV/iodoHoechst are discussed, in view of the potential utility of the ligand as a probe of DNA conformation.     

A 9-base pair deletion in COL1A1 in a lethal variant of osteogenesis imperfecta

A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.     

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.     

Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.

The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.