Search PubMed⌕ Search

Biomedical subjects

C Weissmann

Publications and source records attributed to C Weissmann.

At least 109 records · Page 6Linked to original sources
PubMed1984

Modular structure of the beta-globin and the TK promoters.

Three regions important for transcription, the ATA box, the middle and the distal element, have been identified in the 5'-flanking region of both the rabbit beta-globin and herpes simplex virus thymidine kinase (TK) gene. To determine whether these elements are functionally equivalent, we constructed mosaic promoters containing all combinations of the three regions from both promoters and joined them to the beta-globin transcription unit. In an enhancer-dependent transient expression assay the beta-globin, the TK and all mosaic promoters retaining the beta-globin ATA box were about equally active, however, mosaic promoters with the TK ATA box were 4- to 10-fold less active. We conclude that homologous elements are, in principle, exchangeable and suggest an explanation why certain combinations of elements function poorly.

Animals↗
PubMed1984

A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron.

We constructed rabbit beta-globin genes with deletions in the large intron, extending from the midpoint toward the 5' or 3' splice sites. Analysis of transcripts in transformed HeLa cells showed that six 5' proximal intron nucleotides allowed normal splicing. Correct splicing at the 3' splice site required 12 or more 3' proximal intron nucleotides; optimal efficiency required 24 nucleotides. Remarkably, a mini-intron comprising six 5' and 24 3' intron nucleotides gave no correctly spliced transcripts; extending the miniintron with polyoma or pBR322 fragments to 80 or more nucleotides restored normal splicing. Thus other than in yeast nuclear genes, no specific internal intron sequences appear to be needed but a minimal intron length is important.

Animals↗
PubMed1983

Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli.

We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology. DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E. coli SK1592. Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences. Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques.

Amino Acid Sequence↗
PubMed1983

Structure and expression of cloned murine IFN-alpha genes.

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.

Animals↗
PubMed1983

Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene.

Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells. dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1. SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E. coli (about 18,800) suggests that the former was glycosylated.

Animals↗
PubMed1983

Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene.

Only one of six point mutations of the sequence around one end of the larger of the introns of the rabbit beta-globin gene seriously affects the normal removal of the intron and splicing of the gene. That mutation converts a GT sequence, invariably found at the 5' end of introns, into an AT, which is no longer recognized as a signal for intron removal. Instead, three normally unused (cryptic) sites are used, leading to aberrant gene transcripts. One of the cryptic sites is an exception to the invariable GT sequence.

Animals↗
PubMed1983

Evidence against a scanning model of RNA splicing.

A cloned rabbit beta-globin gene was modified by introducing a DNA fragment containing the 5' splice region of the large intron upstream or downstream of its natural counterpart. Analogous constructions were carried out with the 3' splice region. The genes were linked to SV40 DNA, transiently expressed in HeLa cells and the transcripts analyzed by S1 mapping. In all cases, the splice site further removed from the intron was utilized to the complete exclusion of its counterpart. This finding argues persuasively against a simple scanning model of RNA splicing, in which the splicing enzyme(s) attaches at the 'donor' spliced region and moves along the intron until it encounters the closest 'acceptor' splice region. A model compatible with the currently known facts is presented.

Base Sequence↗
PubMed1983

Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells.

Cloned rabbit beta-globin genes, modified in vitro by restructuring or site-directed mutagenesis, were introduced into mouse 3T6 cells, and the resulting transcripts were analyzed by nuclease S1 mapping. The first 109 bp preceding the cap site sufficed for maximal beta-globin transcription. This segment contained three functionally important regions of the ATA box region; the CCAAT box region; and the -100 region. The latter consists of an imperfect tandemly repeated sequence of 14 bp and 15 bp, both copies of which are required for optimal promoter function. Each of three regions contains two or more nucleotide positions where the introduction of point mutations reduces transcription by at least a factor of 2.

Animals↗
PubMed1982

Structure and expression of human IFN-alpha genes.

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-alpha genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-alpha 1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-beta and IFN-gamma are glycosylated. Because E. coli cells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1 and could be propagated for at least several months.

Base Sequence↗
PubMed1982

Chromosomal location of a human alpha interferon gene family.

To determine the chromosomal location of the human alpha interferon genes, we scored a series of human/rodent somatic cell hybrids for the presence of DNA sequences hybridizing to an alpha 1 interferon DNA probe. The presence of human chromosome 9 in a hybrid correlated with the presence of a family of alpha interferon genes.

Animals↗
PubMed1981

Molecular analysis of the human interferon-alpha gene family.

Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.

Chromosome Mapping↗
PubMed1981

DNA sequences preceding the rabbit beta-globin gene are required for formation in mouse L cells of beta-globin RNA with the correct 5' terminus.

Mouse thymidine kinase-negative (TK-) L cells were transformed with concatenates of cloned herpes simplex virus 1 TK DNA and different rabbit beta-globin DNAs in which the globin genes were preceded by native flanking sequences of 14, 66, 76, 425, and 1500 nucleotides.l In all cases, selection for TK+ cell lines led to a high yield of lines producing 5-1500 mature rabbit beta-globin-specific RNA strands per cell. The 5' termini of the transcripts mapped to (i) the "cap" nucleotide, (ii) positions 42 to 48 nucleotides downstream from the cap site, or (iii) positions in the vector DNA preceding the gene. In the case of the gene with only 14 base pairs of 5' flanking sequence, a high level of rabbit beta-globin RNA was produced, but none of the transcripts had the correct 5' end; most of them originated in the vector moiety. With 66 base pairs of 5' flanking sequence, 5% of the 5' termini were correct, and with 76 or more base pairs, 30-85% were correct. The region between 14 and 66 base pairs preceding the cap site contains the Hogness box and appears to be essential for correct initiation of transcription. The region between 66 and 76 base pairs before the cap site contains a variant of the canonical sequence G-G-C-T-C-A-A-T-C-T found preceding many other genes at a similar location, and this region may modulate the efficiency of transcription. The sequence of 425 nucleotides preceding the rabbit beta-globin gene is reported.

Animals↗
PubMed1981

Target cell specificity of two species of human interferon-alpha produced in Escherichia coli and of hybrid molecules derived from them.

Plasmids containing cDNAs for human interferon (IfN) alpha-1, IFN alpha-2, and several hybrids of the two cDNAs, all joined identically to an Escherichia coli lac promoter fragment gave rise, in E. coli, to fused interferons (fIFNs) that had very different target-cell specificities. fIFN alpha-1 had a lower specific activity on human WISH cells than on bovine MDBK cells, while fIFN alpha-2 showed the opposite behavior. fIFN hybrids with the NH2-proximal half of fIFN alpha-2 behaved qualitatively like fIFN alpha-1, and those with the NH2-proximal half of fIFN alpha-2, behaved like fIFN alpha-2. On mouse L929 cells, fIFN alpha-2 was almost inactive, while fIFN alpha-1 showed relatively high activity. In this case, the fIFN hybrids with the COOH-proximal half of IFN alpha-1 showed activity on mouse cells, while the reciprocal hybrid did not. In many cases, the activity spectrum of the hybrids was very different from that of either parent. We propose that the IFN molecule has either two binding sites or two regions constituting the binding site, one in the COOH- and the other in the NH2-proximal half. The experimental findings can be accounted for if the fits of the two sites to their receptor counterparts on different cell lines are independent of one another.

Animals↗
PubMed1981

Partial mapping of ten genes of the human interferon- alpha family.

HulFN-alpha genes form a family of not less than 9-10 distinct members, at least three of which are arranged in tandem. In one case, two variants, believed to be alleles of the same gene, could be identified by sequence analysis. The three gene sequenced so far are devoid in introns.

Alleles↗
PubMed1980

The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha.

The 12 interferon (IFN)-related sequences detected in a human gene bank fall into not less than eight distinct classes, indicating that there are at least eight IFN-related genes. Most, if not all, of these direct the synthesis of an IFN in Escherichia coli. The sequence of one chromosomal gene and its flanking regions was identical to that deduced for the cDNA corresponding to IFN-alpha l mRNA. No evidence was found for the existence of an intron, in either the coding or the non-coding segments of the gene.

Base Sequence↗