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C Weissmann

Publications and source records attributed to C Weissmann.

215 records · Page 12Linked to original sources
PubMedDate not supplied

Reversed genetics: a new approach to the elucidation of structure--function relationship.

Methods for generating point mutations at predetermined sites of RNA or DNA genomes have been developed. With Qbeta RNA as a template, minus strands were synthesized in vitro in a stepwise, substrate-controlled reaction. The nucleotide analogue N4-hydorxyCMP was introduced in the desired position, the minus strands were completed with the four standard triphosphates and used as templates to synthesize plus strands; about 30% of the progeny plus strands showed a base transition at the position corresponding to the nucleotide analogue. Two mutant RNAs with extracistronic nucleotide substitutions have been generated; one of these was viable, albeit with a reduced propagation rate, while the other was non-infectious. Furthermore, mutants with changes at the initiation codon of the coat cistron were prepared. An analysis of ribosome binding to such mutant RNAs revealed the importance of the A-U-G region for the formation of the initiation complex. With a similar approach applied to the beta-globin complementary DNA (CDNA) plasmid PbetaG, point mutations have been introduced at the positions corresponding to amino acids 121 to 123.

Base Sequence↗
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Secretion of interferon by Bacillus subtilis.

Bacillus subtilis was transformed with a hybrid gene in which the sequence encoding the alpha-amylase signal peptide was joined by a linker to the sequence encoding mature human interferon alpha 2(IFN-alpha 2). The hybrid preprotein was cleaved precisely following the last amino acid of the alpha-amylase signal sequence and was secreted at 0.5--1 mg per liter. IFN-alpha 2, preceded by either one or six amino acids, has the same specific antiviral activity as IFN-alpha 2 itself.

Amino Acid Sequence↗
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Not more than 117 base pairs of 5'-flanking sequence are required for inducible expression of a human IFN-alpha gene.

Interferon genes are usually only expressed after induction. In the accompanying paper we have shown that the accumulation of mRNA after viral induction is due to activation of transcription, rather than to reduction of turnover, and that the regulation of the alpha-interferon (IFN-alpha) gene is mediated by a segment of 5'-flanking region of not more than 700 base pairs (bp). To delineate the sequences required for induction, a set of 5' deletion mutants of the human IFN-alpha 1 gene was constructed and the expression of the truncated genes in mouse L cells was monitored after viral or mock infection. We report that not more than 117 bp of 5'-flanking sequence were required for induced expression of the gene. A purine-rich sequence of 42 bp located immediately downstream of position -117 is highly conserved in all known human alpha-interferon genes.

Animals↗
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The 5'-flanking region of a human IFN-alpha gene mediates viral induction of transcription.

Usually only cells exposed to virus, double-stranded RNA or other inducers synthesize interferon (IFN). Interferon mRNA appears 1-2 h after induction, peaks at 1.5-20 h and decays with a half life of about 30 min. So far, it has not been determined whether induction of interferon is due to transient stabilization of a rapidly turning-over mRNA or to activation of transcription. To clarify this issue we transformed mouse L cells with a hybrid gene in which the 5'-flanking region of the human IFN-alpha 1 gene was followed by the rabbit beta-globin transcription unit. Correctly initiated beta-globin RNA appeared only after viral induction, with the kinetics described for interferon mRNA. Cells transformed with the converse construction, or with the complete rabbit beta-globin gene, constitutively produced correctly initiated transcripts; viral infection decreased the level of transcripts. We conclude that induction acts by activating transcription rather than by reducing turnover, and that the regulatory elements are contained in the 5'-flanking region of the interferon gene.

Animals↗
PubMedDate not supplied

Is sequence conservation in interferons due to selection for functional proteins?

The human alpha-interferon (IFN-alpha) gene family consists of at least 14 potentially functional non-allelic members; the amino acid sequences they encode differ from each other by up to approximately 20% of their residues. Human IFN-beta, which is encoded by a single gene, is distantly related to the IFN-alpha family; it differs in 67% of its residues from IFN-alpha 2. There is considerable evidence that IFN-alpha and -beta compete for the same receptors on their target cells. Comparison of 14 non-allelic human IFN-alpha sequences and the IFN-beta sequence has revealed that 37 of 166 residues are completely conserved and that several of these are arranged in clusters, for example at positions 29-33, 47-50 and 136-150. It is commonly held that evolutionary conservation of amino acids indicates that the residues in question are essential for function. To test this hypothesis in the case of IFNs, we have introduced single site-directed point mutations into the strictly conserved codons 48 and 49 of the IFN-alpha 2 gene which form part of the longest uninterrupted cluster (position 47-50). We report here that the mutant proteins, containing Tyr, Ser and Cys instead of Phe48, or His instead of Gln49, have biological activities indistinguishable from those of wild-type IFN-alpha. In addition, when Glu62, a residue conserved in all known alpha and beta IFNs of man, mouse and cattle, was replaced by Lys, antiviral activity remained unchanged.

Amino Acid Sequence↗
PubMedDate not supplied

Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro.

Introns are excised from full-length transcripts (pre-messenger RNAs) of eukaryotic genes in two steps. First, the pre-mRNA is cleaved at the 5' splice site and a branched (lariat) intermediate is formed. Then, cleavage at the 3' splice site and ligation of the two exons leads to the release of the lariat intron. The intron sequence which accepts the 5' end to form the lariat branch is strictly conserved in yeast, but shows more variation in eukaryotes. To investigate the requirements for branch formation in eukaryotes further, we have studied in vitro splicing of a rabbit globin gene intron with mutations of the normal branch-accepting adenosine nucleotide. We conclude that all four nucleotides can serve as branch acceptors, but that A and C are preferred to G and U in lariat formation. Mutation of the normal A to G or U can lead to an A residue one nucleotide upstream of the normal branch site being used instead. Only branches to A or C participate efficiently in the second splicing step.

Adenine Nucleotides↗