Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping.

A general strategy for cloning and mapping large regions of human DNA with yeast artificial chromosomes (YAC's) is described. It relies on the use of the polymerase chain reaction to detect DNA landmarks called sequence-tagged sites (STS's) within YAC clones. The method was applied to the region of human chromosome 7 containing the cystic fibrosis (CF) gene. Thirty YAC clones from this region were analyzed, and a contig map that spans more than 1,500,000 base pairs was assembled. Individual YAC's as large as 790 kilobase pairs and containing the entire CF gene were constructed in vivo by meiotic recombination in yeast between pairs of overlapping YAC's.

Electrophoretic separations of large DNA molecules by periodic inversion of the electric field.

In gel electrophoresis, nucleic acids and protein-detergent complexes larger than a threshold size all migrate at the same rate. For DNA molecules, this effect can be overcome by the simple procedure of periodically inverting the electric field. Tuning the frequency of the field inversions from 10 to 0.01 hertz, makes it possible to resolve selectively DNA's in the size range 15 to greater than 700 kilobase pairs.

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.

Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Scan of human genome reveals no new Loci under ancient balancing selection.

There has been much speculation as to what role balancing selection has played in evolution. In an attempt to identify regions, such as HLA, at which polymorphism has been maintained in the human population for millions of years, we scanned the human genome for regions of high SNP density. We found 16 regions that, outside of HLA and ABO, are the most highly polymorphic regions yet described; however, evidence for balancing selection at these sites is notably lacking--indeed, whole-genome simulations indicate that our findings are expected under neutrality. We propose that (i) because it is rarely stable, long-term balancing selection is an evolutionary oddity, and (ii) when a balanced polymorphism is ancient in origin, the requirements for detection by means of SNP data alone will rarely be met.

Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae.

CYC1 and sup4 are part of a tightly linked cluster of genes on chromosome X in the yeast Saccharomyces cerevisiae. Using as probes previously cloned fragments containing the CYC1 and sup4 genes, we have identified and cloned the deoxyribonucleic acid (DNA) present between these genes in one strain of yeast. We find that the CYC1 and sup4 genes are approximately 21 kilobases apart. In the same strain, the meiotic map distance is approximately 3.7 centimorgans, for a ratio of 5.6 kilobases per centimorgan in this interval. The physical mapping has allowed unambiguous determination of the orientation of CYC1 and sup4 relative to each other, the centromere, and a nearby transfer ribonucleic acid (tRNA(2Ser)) gene. The spontaneous mutation cyc1-1 inactivates the CYC1 gene as well as the neighboring loci OSM1 and RAD7. We have determined that a cyc1-1-bearing strain lacks approximately 13 kilobases of single-copy DNA from the CYC1-sup4 region, including all of the CYC1 coding information. There is a sequence homologous to the middle-repetitive element Ty1 at or near the breakpoint of the cyc1-1 deletion. We discuss the possibility that Ty elements play a role in the formation of such large, spontaneous deletions, which occur frequently in this region of chromosome X in certain yeast strains.

Inducible nitric oxide synthase activity is essential for inhibition of prostatic tumor growth by interferon-beta gene therapy.

We have previously reported that adenoviral vector-mediated interferon (IFN)-beta gene therapy inhibits orthotopic growth of human prostate cancer cells in nude mice. The purpose of this study was to determine efficacy and mechanisms of this therapy in immune-competent mice. TRAMP-C2Re3 mouse prostate cancer cells infected with 100 multiplicity of infection (MOI) of adenoviral vector encoding for mouse IFN-beta (AdmIFN-beta), but not AdE/1 (a control adenoviral vector), produced approximately 60 ng/10(5) cells/24 h of IFN-beta. The tumorigenicity of AdmIFN-beta-transduced cells was dramatically reduced in the prostates of C57BL/6 mice. A single intratumoral injection of 2 x 10(9) PFU (plaque-forming unit) of AdmIFN-beta inhibited tumor growth by 70% and prolonged survival of tumor-bearing mice. Intriguingly, this AdmIFN-beta therapy did not alter the growth of tumors in inducible nitric oxide synthase (iNOS)-null C57BL/6 mice. Immunohistochemical analysis revealed that treatment of tumors with AdmIFN-beta in wild-type C57BL/6 mice led to increased iNOS expression, decreased microvessel density, decreased cell proliferation, and increased apoptosis. Furthermore, quantitative reverse-transcriptional PCR analysis showed that AdmIFN-beta therapy, in C57BL/6 but not the iNOS-null counterparts, reduced levels of the mRNAs for angiopoietin, basic fibroblast growth factor, matrix metalloproteinase-9, transforming growth factor-beta1, vascular endothelial growth factor (VEGF)-A, and VEGF-B, as well as the antiapoptotic molecule endothelin-1. These data indicated that IFN-beta gene therapy could be effective alternative for the treatment of locally advanced prostate cancer and suggest an obligatory role of NO in IFN-beta antitumoral effects in vivo.

Metaphase and interphase cytogenetics with Alu-PCR-amplified yeast artificial chromosome clones containing the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2.

A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.

Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

Nonsense mutations in essential genes of Saccharomyces cerevisiae.

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae are presented. In order of increasing size, they are chromosomes I, VI, III, IX, V and VIII, comprising 2.49 megabase pairs of DNA. The maps are based on the analysis of an overlapping set of lambda and cosmid clones. Overlaps between adjacent clones were recognized by shared restriction fragments produced by the combined action of EcoRI and HindIII. The average spacing between mapped cleavage sites is 2.6 kb. Five of the six chromosomes were mapped from end to end without discontinuities; a single internal gap remains in the map of chromosome IX. The reported maps span an estimated 97% of the DNA on the six chromosomes; nearly all the missing segments are telomeric. The maps are fully cross-correlated with the previously published SfiI/NotI map of the yeast genome by A. J. Link and M. V. Olson. They have also been cross-correlated with the yeast genetic map at 51 loci.

The yeast cloning vector YEp13 contains a tRNALeu3 gene that can mutate to an amber suppressor.

We have shown that the yeast-Escherichia coli shuttle vector YEp13 contains, as part of its yeast chromosomal segment, a tRNALeu3 gene. We have also isolated and characterized a variant of YEp13 , namely YEp13 -a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13 -a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNALeu3 anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNALeu3 gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNALeu3 gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53 . We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNALeu3 genes has revealed considerable sequence homology in the immediate 5'-flanking regions of these genes.

Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation.

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.