Fragments of Aspergillus nidulans DNA obtained after digestion with EcoRI, BamHI and HindIII endonucleases were cloned in Escherichia coli in plasmid pBR322. These gene banks were used for transformation of 15 E. coli auxotrophic mutants and in 5 cases prototrophic clones containing recombinant plasmids were selected. Three different recombinant plasmids conferring prototrophy to pyrF, proAB and argIF mutants were analyzed. Hybridization experiments indicated that in two of these plasmids the inserted fragment of DNA hybridized not only to the A. nidulans but also to the E. coli DNA.

Transfection of embryo fibroblasts by a human ras oncogene does not convert them into tumour cells unless the fibroblasts are established and immortalized before transfection. The embryo fibroblasts become tumorigenic if a second oncogene such as a viral or cellular myc gene or the gene for the polyoma large-T antigen is introduced together with the ras gene.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

The location of cis-acting regulatory sequences within the long terminal repeat (LTR) of the human T cell lymphotropic virus type III (HTLV-III/LAV) was determined. An enhancer element capable of increasing the rate of transcription from a heterologous promoter, irrespective of distance and orientation, is located between nucleotides -137 and -17 (cap site +1). The promoter sequences present near the TATA box respond to heterologous enhancers. The sequences present between nucleotides -17 and +80 are responsive to HTLV-III-associated trans-acting regulatory factors. Activation of these sequences by the viral regulatory factors requires the presence of a functional enhancer. The enhancer requirement is nonspecific, as the enhancer sequences of RSV, HTLV-I, and SV40 can functionally replace the HTLV-III enhancer. These findings define a new type of regulatory element, provide insight into the mechanisms that regulate HTLV-III gene expression, and may help to explain the effects of this virus on infected cells.

Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells. Because of their high copy number, the replication of such plasmids has been described as 'relaxed', even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers and ColE1 copy-number mutants have been characterised. No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells. Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells. Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number. Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells. Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised.

The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA(+)) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.