Mutations In Essential Genes Research Articles

Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.

Efficient expression of small RNA polymerase III genes from a novel Simian virus 40 vector and their effect on viral gene expression

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

Charons 36 to 40: multi enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers.

New phage lambda based cloning vectors, Charons 36-40, have been constructed which allow cloning of large (up to 24 kb) DNA fragments with up to sixteen cloning enzymes. Several of these could not be used previously with lambda vectors. Clones produced with these vectors can be propagated under recombination deficient conditions. A novel polystuffer method has been developed that permits vector arms to be purified by simple precipitation and which allows reliable identification of clones that have reincorporated any part of the stuffer. Three of the vectors are available with amber mutations in essential genes.

Genetic organization of the region around UNC-15 (I), a gene affecting paramyosin in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans mutants in the gene unc-15 (I) affect the muscle protein paramyosin (Waterston, Fishpool and Brenner 1977). We have characterized 20 ethyl methanesulfonate-induced mutations in essential genes closely linked to unc-15. These lethals defined 16 new complementation groups. In the 0.65 map-unit interval around unc-15 defined by dpy-14 and unc-56, seven newly identified genes have been mapped relative to five existing genes. At present, the average distance between genes in this region is approximately 0.05 map units. Two genes, unc-15 and unc-13, are only 0.025 map units apart. Partial fine-structures maps of alleles of these two genes have been constructed. This analysis of unc-15 and genes adjacent to it is the first in a series of genetic and biochemical studies directed towards understanding the control of unc-15 expression.

Correction and refinement of the genetic map of bacteriophage Mu

Three hundred sixty-one amber mutations in essential genes of bacteriophage Mu have been assigned to 1 of 76 deletion groups on the basis of results of deletion mapping by marker rescue from 90 λpMu transducing phage. The original order of previously defined cistrons determined from prophage deletion mapping has been confirmed with the exception that cistron O has been deleted and cistron H has been found to map between cistrons E and F rather than between cistrons G and I. Mapping of mutations in the new cistrons T, U, V, W. and Y ( Howe et al., 1979a, Virology 93, 303–319 ) has produced the following cistron order: ABClysDEHFGITJKLMYNPQVWRSU.

Isolation of mutations defining five new cistrons essential for development of bacteriophage Mu

Three hundred new mutant strains of bacteriophage Mu with amber mutations in essential genes were isolated and characterized. Measurement of complementation between these new strains and strains carrying mutations in previously identified complementation groups revealed the existence of five new complementation groups: T, U, V, W, and Y. In general, mixed infection of cells by phage carrying mutations in different cistrons resulted in the production of a normal burst of 50 to 200 phage per cell; however, mixed infection with phage carrying mutations in certain pairs of adjacent cistrons gave a 10- to 20-fold lower burst of only 3 to 30 phage per cell. Cistron pairs which showed this reduced complementation were D- E, H- F, J- K, Y- N, Q- V, W- R, and S- U. In addition, mutations assigned to cistron I fell into three groups on the basis of their complementation behavior; phage carrying mutations in one group located at one end of the cistron showed weak complementation with phage carrying mutations in a second group at the opposite end of the cistron, but phage carrying mutations in the third group located between the first two groups showed no complementation with members of any of the three groups. The complementation analysis also showed that mutations previously assigned to separate complementation groups O and P really belong to a single complementation group P.

Essential genes in the metB-malB region of Escherichia coli K-12.

We isolated 25 independent mutants that are deficient in essential genes located in the metB-malB region of the Escherichia coli chromosome. The mutations were mapped within this region by using several F' factors and were also classified into 11 cistrons by complementation testing. There is clustering of mutations in essential genes within the metB-malB region.

Temperature-sensitive nonsense mutations in essential genes of Escherichia coli.

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.

Localized mutagenesis of any specific small region of the bacterial chromosome.

A method, which we call localized mutagenesis, is described for the isolation of temperature-sensitive and other types of mutations in any specific small region (about 1%) of the bacterial chromosome. The principle of this method is to mutate the transducing DNA rather than the bacterial DNA. One can select for the introduction of this mutated DNA into any particular region of the bacterial chromosome by transducing an auxotrophic marker in that region to prototrophy, thereby introducing new mutations in the neighborhood. We have used this method to isolate many different temperature-sensitive mutations in genes of unknown function in particular regions of the chromosome. Since the method is very simple, it can be used to saturate any region of the map with mutations in essential genes, or for various types of genetic manipulations. Although we have used hydroxylamine-mutagenized phage P22 and Salmonella typhimurium, the method should be applicable to other mutagens and bacteria and transducing phage.