Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.     

Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene.

cdc28, one of several genes required for cell division in the yeast Saccharomyces cerevisiae, has been isolated on recombinant plasmids. A recombinant plasmid pool containing the entire yeast genome was constructed by partial digestion of yeast DNA with the four-base recognition restriction endonuclease Sau3A to give the equivalent of random fragments, size selection on sucrose gradients, and introduction of the fragments into the yeast vector YRp7 by use of the homology of Sau3A ends with those generated in the vector by cleavage with BamHI. Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus, confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants.     

Isolation of the centromere-linked CDC10 gene by complementation in yeast.

A hybrid plasmid colony bank was constructed in Escherichia coli using the E. coli-Saccharomyces cerevisiae shuttle vector pLC544 and randomly sheared segments of yeast DNA. By transformation with a hybrid plasmid DNA pool from this collection and complementation of a temperature-sensitive cdc10 mutation in yeast, a plasmid was isolated that carries 8 kilobase pairs of DNA around the chromosome III centromere-linked CDC10 locus. This DNA segment overlaps a larger region of DNA (40 kilobase pairs) previously identified to be around the LEU2 locus on chromosome III [Chinault, A.C. & Carbon, J. (1979) Gene 5, 111-126] and physically establishes the directionality of the cloned DNA sequences with respect to the genetic map and the centromere. In the leu2-cdc10 interval, the relationship between physical distance on the DNA and genetic distance as measured by recombinational frequencies is about 3 kilobase pairs per centimorgan.     

Consistent association between sigma elements and tRNA genes in yeast.

Sigma is a recently described family of transposable elements in yeast (Saccharomyces cerevisiae). The most striking feature of the seven sigma elements that have been previously identified is that all are located 16-18 base pairs upstream from tRNA-encoding regions. Because these cases were all encountered in the process of studying specific tRNA genes, the full extent of the association between sigma elements and tRNA genes could not be assessed. In this paper, we report a more global characterization of the sigma family in a typical laboratory yeast strain: of the 30 copies of sigma that we estimate to be present in the haploid genome, we have cloned and analyzed 25 loci. Although in two cases a pair of sigma elements were found within several kilobases of each other, the majority occur as individual elements at widely dispersed sites. Moreover, in all 25 cases analyzed, the sigma elements are closely associated with tRNA genes. Thus, the sigma transposable element has been shown to have an absolute association with another gene family.     

Temperature-sensitive mutations in the yeast DNA polymerase I gene.

Seven mutations that yield thermolabile DNA polymerases have been isolated in the DNA polymerase I gene, POL1, of Saccharomyces cerevisiae. Strains carrying the mutant genes are temperature sensitive for growth. The pol1 mutants were identified by a method that has general applicability for identification of both temperature-sensitive and null mutations. A plasmid containing a mutagenized pol1 gene was transformed into a strain in which the only functional copy of the POL1 gene was carried on an unstable plasmid. The genes conferring temperature-sensitive growth were detected after elimination of the unstable plasmid containing the wild-type gene. DNA polymerase I isolated from each of the mutants is defective at both 23 degrees C and 36 degrees C. DNA synthesis is deficient in vivo at 36 degrees C in all the mutants, while RNA synthesis is normal in all but one of the mutants. The terminal phenotype of pol1 temperature-sensitive mutants is dumbbell-shaped cells in which the nucleus has migrated to, but apparently not entered, the isthmus separating the mother and the daughter. The POL1 gene is located on chromosome XIV approximately 2 centimorgans away from met4.     

Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system, referred to here as automatic subcloning. A simple method has been developed for the conversion of any plasmid into a phage lambda cDNA cloning vector with automatic subcloning capability. cDNA libraries constructed in these vectors were used to isolate genes from humans and Arabidopsis thaliana by complementation of yeast and bacterial mutations, respectively.     

Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown.     

Selection of functional cDNAs by complementation in yeast.

Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1%. The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.     

Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.     

Screening for MEN1 tumor suppressor gene mutations in sporadic pituitary tumors

The molecular pathogenesis of the majority of sporadic pituitary tumors is largely unknown. Pituitary adenomas can develop sporadically or as a part of multiple endocrine neoplasia type 1 (MEN1). The MEN1 is thought to be a tumor suppressor gene based on loss of heterozygosity (LOH) for polymorphic markers on 11q13 in tumors of the pancreas, parathyroid, and pituitary. Most patients with familial and sporadic MEN1 carry germ-line mutations in the MEN1 gene. Two previous studies and recently a third one have analyzed mutations by sequencing the MEN1 gene in sporadic pituitary tumors but yielded conflicting results. This study was to investigate and clarify the potential role of MEN1 mutations, in sporadic pituitary adenomas. First, we examined 59 sporadic pituitary adenomas by analyzing LOH on 11q13 in the MEN1 minimal interval with microsatellite analysis. We found 3 tumors with LOH in 1 to 4 polymorphic markers in the MEN1 region. Sequencing analysis did not reveal any mutations in the coding region of the MEN1 gene. However, we found 3 polymorphisms, one of which was a novel CAC to CAT transition encoding His433His, in exon 9. The data show that while LOH occurs in some sporadic pituitary tumors, inactivating mutations of the tumor suppressor gene MEN1 are rare. These results also suggest there may be another additional tumor suppressor gene at this locus which is involved in the pathogenesis of sporadic pituitary neoplasms.     

Chloroplast gene sequences and the study of plant evolution.

A large body of sequence data has accumulated for the chloroplast-encoded gene ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) as the result of a cooperative effort involving many laboratories. The data span all seed plants, including most major lineages from the angiosperms, and as such they provide an unprecedented opportunity to study plant evolutionary history. The full analysis of this large data set poses many problems and opportunities for plant evolutionary biologists and for biostatisticians.     

Chloroplast tRNA gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA (UCC) and tRNA (UCU)

The nucleotide sequences of tobacco chloroplast genes for tRNA^Gly (UCC) and tRNA^Arg (UCU) have been determined. The tRNA^Gly gene has a 691-base-pair intron located in the D stem while the tRNA^Arg gene does not have any intron. The tRNA^Gly and tRNA^Arg genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA^Gly gene is transcribed as a 900-base precursor RNA molecule. The tRNA^Gly and tRNA^Arg deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA^Gly (UCC) and phage T4 tRNA^Arg (UCU), respectively.     

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.     

Functional consequences of integrin gene mutations in mice

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of mice lacking the integrin ligands.     

Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast.

In a screen designed to isolate human cDNAs that complement a yeast G2 phase checkpoint mutation (mec1), we isolated a cDNA homologous to the Saccharomyces cerevisiae CDC34 gene. The human CDC34 cDNA can functionally substitute for the yeast CDC34 gene and represents a mammalian homolog of the group of yeast genes required for the late G1-->S phase transition. The human CDC34 gene is expressed in multiple cell lines as a unique species and Southern blot analysis reveals evidence for a single gene that is highly conserved in higher eukaryotes. The human gene is located on the far telomeric region of 19p13.3 in a location that defines a region of homology between human chromosome 19p and mouse chromosome 11.