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C Yanofsky

Publications and source records attributed to C Yanofsky.

At least 109 records · Page 6Linked to original sources
PubMed1988

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.

DNA, Fungal↗
PubMed1988

Molecular analysis of a Neurospora crassa gene expressed during conidiation.

The asexual developmental pathway in the life cycle of the filamentous fungus Neurospora crassa culminates in the formation of spores called conidia. Several clones of genomic Neurospora DNA have been isolated that correspond to mRNA species expressed during conidiation and not during mycelial growth (V. Berlin and C. Yanofsky, Mol. Cell. Biol. 5:849-855, 1985). In this paper we describe the characterization of one of these clones, named pCon-10a. This clone contains two genes, con-10 and con-13, which are induced coordinately during the later stages of conidiation. The two genes are separated by 1.4 kilobases of DNA; they are located on linkage group IV and are transcribed from the same strand of DNA. The molecular organization and sequence of one of these genes, con-10, and its flanking regions are presented. Full-length cDNA clones for con-10 also were isolated and sequenced, and transcription-initiation and polyadenylation sites were defined. The con-10 gene contains an open reading frame interrupted by two small introns and encodes an 86-amino-acid residue polypeptide that is both hydrophilic and weakly acidic. Expression of the con-10 gene in various mutants defective at different stages of conidiation indicates that it plays a role after aerial hyphal development. Possible functions, organization, and regulation of conidiation-specific genes are discussed.

Base Sequence↗
PubMed1987

Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide.

The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains. In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments. The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA. Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi. To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation. We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein. In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2. The fusion proteins have normal substrate affinities. Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites. This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.

Base Sequence↗
PubMed1987

Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex.

Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.

Bacterial Proteins↗
PubMed1987

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.

Bacterial Proteins↗
PubMed1987

Crucial role of the connecting region joining the two functional domains of yeast tryptophan synthetase.

We constructed a hybrid plasmid expressing yeast tryptophan synthetase in Escherichia coli. Several deletion variants lacking the A or B domains of this polypeptide (recognized by their homology to the alpha and beta subunits of prokaryotic tryptophan synthetase) showed no enzymatic activity and failed to substitute for the corresponding E. coli subunits. To examine the role of a presumed interdomain connecting region in the yeast enzyme, we constructed a variant lacking 18 amino acids in that region. The variant polypeptide was completely inactive. Replacing 14 of the 18 missing amino acids with a segment having a different sequence partially restored activity. A spontaneous revertant was characterized and shown to have a duplication of 16 amino acid residues in this region; the activity of the duplication polypeptide was better than that of the 14-residue replacement. If confirmed by additional studies, our finding that the length of the connecting region is more critical than its sequence has implications for understanding the origin of gene fusions during evolution as well as for designing artificial fusions.

Amino Acid Sequence↗
PubMed1987

Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells.

We have produced a series of hybrid IgG1.IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differences between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse gamma 1 and gamma 2a gene segments. Crossovers occurred throughout these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglobulins have identical antigen combining sites specific for the fluorescent hapten epsilon-dansyl-L-lysine. Protein A binding was used as a probe of the structural integrity of the Fc portion of these variant antibodies. Differences in protein A binding between IgG1 and IgG2a appear to be due to amino acid differences at positions 252 (Thr----Met) and 254 (Thr----Ser) of the heavy chain (EU numbering).

Amino Acid Sequence↗
PubMed1987

Detection of transcription-pausing in vivo in the trp operon leader region.

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.

Escherichia coli↗
PubMed1986

High level production and rapid purification of the E. coli trp repressor.

Two small, multicopy, expression plasmids were constructed that permit convenient insertion of trpR, the structural gene for the trp repressor of Escherichia coli, with its natural ribosome binding site or adjacent to the ribosome binding site for the trp leader peptide. In these plasmids trpR is positioned between the strong regulated tac promoter and the rpoC transcription terminator. IPTG induction of lacIq strains bearing these plasmids results in the production of 25-50% of the soluble cell protein as trp repressor. Mutant and wild type repressors overproduced in this manner have been purified by simple procedures.

Apoproteins↗
PubMed1986

Efficient cloning of genes of Neurospora crassa.

We have constructed a genomic library of Neurospora crassa DNA in a cosmid vector that contains the dominant selectable marker for benomyl resistance. The library is arranged to permit the rapid cloning of Neurospora genes by either sib-selection or colony-hybridization protocols. Detailed procedures for the uses of the library are described. By use of these procedures, a modest number of unrelated genes have been isolated. The cloning of trp-3, the structural gene for the multifunctional enzyme tryptophan synthetase (tryptophan synthase, EC 4.2.1.20), is reported in detail; its identity was verified by restriction fragment length polymorphism mapping. The strategies described in this paper should be of use in the cloning of any gene of Neurospora, as well as genes of other lower eukaryotes.

Journal Article↗
PubMed1986

Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12.

Recent studies have suggested that expression of the tryptophanase (tna) operon of Escherichia coli is subject to transcription termination-antitermination control (V. Stewart and C. Yanofsky, J. Bacteriol. 164:731-740, 1985). In vivo studies have indicated that the transcribed leader region, tnaL, contains a site or sites of rho-dependent transcription termination (rho is the polypeptide product of the gene rho). We now report direct in vitro evidence that tnaL contains rho-dependent termination sites. In vivo termination appeared to occur at the rho-dependent termination sites identified in vitro. Transcription pausing analyses correlated sites of pausing in tnaL with sites of rho-dependent termination.

Base Sequence↗
PubMed1986

Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon.

Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.

Amino Acid Sequence↗
PubMed1986

Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons.

The trp operon regulatory region of Bacillus pumilus was cloned and sequenced. The cloned B. pumilus trp promoter-leader region functioned in Bacillus subtilis to express the adjacent leukocyte interferon A gene on a multicopy transcriptional fusion plasmid, pBpIFI. In strains carrying this plasmid, anthranilate synthetase levels were elevated, possible due to titration of a B. subtilis trp regulatory factor by multiple copies of the transcript of the plasmid-borne B. pumilus trp leader region. The B. pumilus trp promoter was recognized efficiently in vitro by B. subtilis sigma 43 RNA polymerase. Approximately 12% of the transcripts produced in vitro terminated in the leader region immediately following synthesis of a transcript structure resembling rho-independent terminators of enteric bacteria. An analogous terminator exists in the B. subtilis trp leader transcript. Nucleotide sequence comparison of the B. pumilus and B. subtilis trp leader regions revealed conservation of these and other sequences that could form transcript secondary structures postulated to regulate transcription termination in B. subtilis (H. Shimotsu, M.I. Kuroda, C. Yanofsky, and D.J. Henner, J. Bacteriol. 166:461-471, 1986). We propose that two elements implicated in B. subtilis trp operon regulation are conserved in the related organism B. pumilus: alternative transcription antiterminator and terminator structures in the leader transcript, and a trans-acting factor present in limiting amounts that is required for transcription termination in the leader region.

Anthranilate Synthase↗
PubMed1986

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Amino Acid Sequence↗
PubMed1985

Analysis of the requirements for transcription pausing in the tryptophan operon.

RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide. Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon. We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro. A model is presented that accounts for many of the findings obtained in this and other pausing studies.

Base Sequence↗
PubMed1985

Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa.

We have cloned the his-3 gene of Neurospora crassa and determined its nucleotide sequence. The gene specifies a protein of 863 amino acids (aa) and contains a 59-bp intron which interrupts aa 800, a proline residue. The 5' end of the his-3 transcript is heterogeneous with major starts 122 and 124 bp upstream from the start codon. There are three possible polyadenylation sites, 119, 120 and 121 bp after the UAA stop codon. The protein shows two regions of homology to the yeast HIS4 gene which correspond to the hisIE and hisD genes of Escherichia coli and Salmonella typhimurium. Northern analysis shows that the level of his-3 mRNA increases severalfold in cultures subjected to histidine starvation.

Base Sequence↗