Charles Yanofsky

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.

Nonsense codons and polarity in the tryptophan operon

Abstract Amber and ochre mutants and mutants with cross-reacting material for all five genes of the tryptophan operon have been isolated and characterized. Co-ordinate enzyme formation was examined under repression conditions with a repressordeficient stock of each mutant. It was found that alterations resulting in the production of cross-reacting material had little or no effect on relative rates of synthesis of tryptophan enzymes, whereas amber and ochre nonsense alterations invariably had polar effects—they reduced the relative rates of synthesis of all proteins specified by genes in the operon more distal from the operator region than the gene with the nonsense mutation. Nonsense alterations on the side of a gene close to the operator region had a more pronounced effect on the synthesis of proteins specified by more distal genes in the operon than alterations near the other end of the gene. Amber and ochre alterations had equivalent effects on enzyme formation. Represser-deficient stocks were prepared with nonsense alterations in two different genes in the operon. Each nonsense alteration acted independently in reducing the relative rate of synthesis of the protein specified by the last gene of the operon. Repressor-deficient stocks with two nonsense alterations in the same gene behaved in co-ordinate enzyme production studies as if only one nonsense alteration were present. Suppressor genes relieved the polar effect, approximately to the same extent as they permitted synthesis of the protein specified by the gene with the nonsense alteration. Under de-repression conditions, nonsense alterations in the first gene of the operon had the most profound effect on enzyme formation. An interpretation of these findings is presented which suggests that ribosomes can only become attached at the operator end of the polycistronic tryptophan messenger and that ribosomes leave the messenger in regions between an introduced nonsense codonand the start of the next message. Polarity would result from the reduction of the number of active ribosomes on the messenger following the encounter with the nonsense codon and the subsequent untranslatable region.

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.

Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa.

Carotenoid biosynthesis is regulated by blue light during growth of Neurospora crassa mycelia. We have cloned the al-1 gene of N. crassa encoding the carotenoid-biosynthetic enzyme phytoene dehydrogenase and present an analysis of its structure and regulation. The gene encodes a 595-residue polypeptide that shows homology to two procaryotic carotenoid dehydrogenases. RNA measurements showed that the level of al-1 mRNA increased over 70-fold in photoinduced mycelia. Transcription run-on studies indicated that the al-1 gene was regulated at the level of initiation of transcription in response to photoinduction. The photoinduced increase of al-1 mRNA levels was not observed in two Neurospora mutants defective in all physiological photoresponses. Analysis of cosmid containing al-1 and of a translocation strain with a breakpoint within al-1 indicated that al-1 transcription proceeds towards the centromere of linkage group I of N. crassa.

Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli

Abstract The relationship between gene sequence and the sequence of biochemical reactions in the biosynthesis of tryptophan was investigated in the K12 strain of Escherichia coli. Transduction with phage P1kc was employed for genetic mapping. All the tryptophan auxotrophs examined were found to be mutant in a small region of genetic material (>80% joint transduction of the most distal markers). One strain with a block in the synthesis of several aromatic compounds, including tryptophan, was found to be mutant at a site weakly linked to the other tryp genes. Two cysteine auxotrophs were also mutant at sites linked to the tryp region. The transduction tests employed suggested the following order of the tryp genes and the cysteine markers: cys … tryp 4. tryp 3. tryp 1. tryp 2. This order differs from the gene order established with the corresponding mutants of Salmonella typhimurium only with respect to the last two tryp genes, tryp 1 and tryp 2. However, since the proteins controlled by these genes are both involved in the terminal reaction in tryptophan synthesis, and thus cannot be assigned a biosynthetic order, the results obtained indicate that the clustered tryp genes of E. coli are arranged in the same relative order as the steps in the biosynthesis of tryptophan which they control. An examination of the category of tryptophan auxotrophs which acquired a requirement for tryptophan simultaneously with mutation to resistance to phage T1 suggested that these strains arose as a result of deletions including the V1r locus and one or more tryp genes. It was found that in strain B, where mutations of this type are fairly frequent, most of the V1r tryp mutants lack all the clustered tryp genes. In strain K12, where V1r tryp mutants are rare among V1r isolates, the few strains obtained did not lack all the clustered tryp genes. Studies with hybrids formed between strains K12 and B suggested that genic material at or near the tryp region in the two strains determines the type of mutation pattern when cells are exposed to T1. The deletion from one V1rtryp mutant was transferred by transduction and 60% of the phage carrying the tryp 4 marker were found to carry the entire tryp region and thus were capable of converting tryp deletion mutants to tryptophan-independent strains.

Transcription Attenuation: Once Viewed as a Novel Regulatory Strategy

The views expressed in this feature do not necessarily represent the views of the journal or of ASM . Regulation by transcription termination/antitermination, transcription attenuation, is a commonly used strategy. It is most often based on selective formation of either of two alternative base-

Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium.

We have obtained the entire nucleotide sequence of the penultimate gene of the tryptophan operon, trpB, in Escherichia coli and Salmonella typhimurium. The amino acid sequence deduced for the E. coli gene product is in agreement with earlier, fragmentary protein sequence results. The trpB nucleotide sequences for the two bacterial species are perfectly colinear and show 85% identity. Most of the nucleotide differences found are without consequence for the amino acid sequence, which shows greater than 96% identity. The degree of conservation of both the nucleotide and amino acid sequences is significantly greater than for trpA, the adjacent gene encoding the other subunit of the same enzyme. When synonymous third codon position nucleotide differences are examined, they seem to be distributed at random throughout trpB and trpA, except for one completely conserved 66 basepair long region within trpB.

Transcription of the tryptophan operon in polarity mutants of Escherichia coli. I. Characterization of the tryptophan messenger RNA of polar mutants.

Amber, ochre, and frame-shift polarity mutants of the tryptophan operon of Escherichia coli were examined in transcription studies to determine whether translational polarity was associated with an alteration of the tryp -mRNA population. Tryp -mRNA was isolated from bacterial cultures following a shift from repression to derepression conditions. The cultures were pulse-labeled with [ 3 H]uridine at some period soon after the initiation of derepression. Total tryp -mRNA and tryp -mRNA regions corresponding to different segments of the operon were measured by hybridizing with DNA from o80 transducing phages carrying different segments of the operon. It was found that strong polarity mutants generally contained less total tryp -mRNA than the identically treated wild-type strain or missense mutant. This result was most evident with strong polarity mutants with alterations in the E gene, but the data obtained are suggestive for strong polarity mutants with alterations in the other genes of the operon. Specific hybridization studies demonstrated that the tryp -mRNA of strong polarity mutants was deficient in the mRNA regions corresponding to the genes of the operon on the operator-distal side of the mutated gene. In agreement with this finding, sucrose gradient sedimentation profiles of the tryp -mRNA of strong polarity mutants indicated that the majority of the tryp -mRNA molecules of each mutant were smaller than normal. It was also shown that the size of the predominant tryp -mRNA species in different mutants increased in relation to the genetic distance from the beginning of the first gene of the operon to the site of the mutation. Estimates of the relative numbers of normal length and smaller-than-normal tryp -mRNA molecules in strong and weak polarity mutants gave values consistent with the conclusion that the extent of translational polarity in any mutant approximates the relative extent of representation of different operon regions in the tryp -mRNA population.

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

SUMMARY The T-box mechanism is a common regulatory strategy used for modulating the expression of genes of amino acid metabolism-related operons in gram-positive bacteria, especially members of the Firmicutes. T-box regulation is usually based on a transcription attenuation mechanism in which an interaction between a specific uncharged tRNA and the 5′ region of the transcript stabilizes an antiterminator structure in preference to a terminator structure, thereby preventing transcription termination. Although single T-box regulatory elements are common, double or triple T-box arrangements are also observed, expanding the regulatory range of these elements. In the present study, we predict the functional implications of T-box regulation in genes encoding aminoacyl-tRNA synthetases, proteins of amino acid biosynthetic pathways, transporters, and regulatory proteins. We also consider the global impact of the use of this regulatory mechanism on cell physiology. Novel biochemical relationships between regulated genes and their corresponding metabolic pathways were revealed. Some of the genes identified, such as the quorum-sensing gene luxS, in members of the Lactobacillaceae were not previously predicted to be regulated by the T-box mechanism. Our analyses also predict an imbalance in tRNA sensing during the regulation of operons containing multiple aminoacyl-tRNA synthetase genes or biosynthetic genes involved in pathways common to more than one amino acid. Based on the distribution of T-box regulatory elements, we propose that this regulatory mechanism originated in a common ancestor of members of the Firmicutes, Chloroflexi, Deinococcus-Thermus group, and Actinobacteria and was transferred into the Deltaproteobacteria by horizontal gene transfer.

[107] Enzymes involved in the biosynthesis of tryptophan

Publisher Summary When a group of tryptophan auxotrophs are obtained after treatment of a tryptophan-nonrequiring microorganism with a mutagenic agent, many biochemically different mutant types are usually represented. Extracts of most of these mutants are unable to catalyze the formation of tryptophan from its precursor, anthranilic acid. One of the mutant extracts contains the enzymes required to convert the substrate supplied, anthranilic acid, to some intermediate, but lacks one of the enzymes required to further metabolize the intermediate. The extract of the second mutant supplies this deficient enzyme and allows the complete conversion to take place. Use of the proper mutant extract alone, therefore, with the appropriate substrate, permits the formation and the accumulation of an intermediate which otherwise might not have been detected. Thus mutant extracts provide a ready source of specific enzymatic activities, essentially free of the next sequentiallyrelated enzymatic activity. The chapter describes the formation, assay method, purification procedure, and properties of anthranilic deoxyribulotide. The assay procedure is based on the decrease of the characteristic fluorescence due to anthranilie acid during the course of the enzymatic reaction. With ATP and ribose-5-P as substrates for PP-ribose-P synthesis, anthranilie acid utilization is proportional to enzyme concentration over a range of enzyme dilutions. The same is true with PPribose- P as substrate.