Variation in the polyadenylylation site of bovine prolactin mRNA.

The poly(A) site of bovine prolactin (bPRL) mRNA was examined by phased priming of cDNA synthesis with oligodeoxynucleotides of the general sequence d(pT8-N-N'). The existence of multiple poly(A)-adjacent sequences in bPRL mRNA was indicated by the production of specific chain-termination fragments with at least three d(pT8-N-N') sequences. Comparison of the sequence bands produced by initiation of cDNA synthesis on the bPRL mRNA template with d(pT8-A-G), d(pT8-G-A), and d(pT8-C-G) revealed a shifted pattern of identical fragments. The shift in position of related sequence bands on the gel suggested that the difference in length of the three major bPRL mRNA species occurred within a span of 12 nucleotides. Sequence analysis conducted with the three d(pT8-N-N') primers gave identical nucleotide sequences for the 3' noncoding region of the bPRL mRNA species and suggested that the mRNA molecules were heterogeneous in length. The existence of multiple poly(A) sites was confirmed by determination of the nucleotide sequence of bPRL cDNA clones containing two of the major poly(A)-adjacent sequences predicted by the oligodeoxynucleotide primers. The mRNA molecules containing these multiple poly(A)-addition sites were shown to be present in the bPRL mRNA obtained from a single pituitary gland. The variation in the poly(A) junction of bPRL mRNA may be a reflection of the processing events at the 3' terminus of mRNAs.     

Endonuclease S1-sensitive site in chicken pro-alpha 2(I) collagen 5'' flanking gene region.

A site that is preferentially cleaved by the single-strand-specific endonuclease from Aspergillus oryzae was located in vitro 180 base pairs upstream from the 5' end of the chicken pro-alpha 2(I) collagen gene. It is found in supercoiled plasmids with a negative superhelical density of -0.024 or more but not in linear DNA molecules. The nuclease S1 sensitivity is retained in plasmids containing genomic fragments extending from position +8 to -285 (where +1 is the first transcribed base) and from -147 to -351 and also in a 5.7-kilobase EcoRI fragment that extends 1.6 kilobases 5' and 4.1 kilobases 3' to the 5' end of the gene. Analysis at the nucleotide level on a DNA sequence gel places the site at -181 to -182 on the sense strand and at -182 to -184 and -192 to -195 on the nonsense strand. These sites lie within a stretch of 42 pyrimidines interrupted by a single guanine and within the sequence T-C-C-C-T-C-C-C-T-T-C-C-T-C-C-C-T-C-C-C-T.     

Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.

To determine the importance of the third alpha-helix in bovine growth hormone (bGH) relative to growth-related biological activities, the following experimental approach was used: (i) mutagenesis of helix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies of wild-type and mutated bGH; and (iv) expression of the mutated gene in the transgenic mouse. An altered bGH gene (pBGH10 delta 6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGH10 delta 6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growth-suppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule.     

Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5'-flanking region of the bovine parathyroid hormone gene

Receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5'-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning -700 to +50 bp with 200 micrograms cytosolic protein gave 288 +/- 63% (mean +/- S.D.) of binding in the absence of protein. In contrast, there was no significant reaction with the -1350 to -700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the -700 to +50 bp fragment. The receptor-binding site was further defined to -700 to -100 bp as deletion of the -100 to +50 bp did not reduce receptor binding. Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50,000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the -700 to +50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. (ABSTRACT TRUNCATED AT 250 WORDS)     

DNA rearrangements in human follicular lymphoma can involve the 5'' or the 3'' region of the bcl-2 gene.

In most human follicular lymphomas, the chromosome translocation t(14;18) occurs within two breakpoint clustering regions on chromosome 18, the major one at the 3' untranslated region of the bcl-2 gene and the minor one at 3' of the gene. Analysis of a panel of follicular lymphoma DNAs using probes for the first exon of the bcl-2 gene indicates that DNA rearrangements may also occur 5' to the involved bcl-2 gene. In this case the IgH locus and the bcl-2 gene are found in the order 3' C gamma S gamma/mu JH 5'::5' bcl-2 3' (where C = constant, S = switch, and JH = joining segment of the heavy chain locus), suggesting that an inversion also occurred during the translocation process. The coding regions of the bcl-2 gene, however, are left intact in all cases of follicular lymphoma studied to date.     

Human U1 RNA genes contain an unusually sensitive nuclease S1 cleavage site within the conserved 3'' flanking region.

We find that the cloned DNAs of human U1 small nuclear RNA genes contain two nuclease S1-sensitive sites, one about 1.8 kilobases downstream of the U1 RNA coding region and the other around 0.3 kilobase upstream. The downstream site is unusually sensitive to the nuclease, being cleaved in both linear and negatively supercoiled DNAs. The extent of cleavage at this site is enhanced at lower pH and reduced concentrations of NaCl; the effects of salt are more apparent on linear than supercoiled DNAs. The nuclease S1 sensitivity of this downstream site is dependent on the presence of the sequence (dC-dT)n X (dA-dG)n, where n = 15-25. (One gene with n = 5 is resistant to nuclease S1 cleavage in this region.) In contrast, the nuclease S1 site upstream of the coding region is cleaved only when the DNA is supercoiled. This site also has a homopyrimidine X homopurine bias in the DNA strands, but the sequence is less regular. In the course of these studies, we detected several discrepancies between our restriction maps of some U1 RNA genes and those published by others. Our maps demonstrate that all seven cloned human U1 RNA genes are very similar in sequence for as much as 2.3 kilobases downstream of the U1 RNA coding region.