Isolation and characterization of Staphylococcus aureus strains from a Paso del Norte dairy.

The primary purpose of this study was to determine if methicillin-resistant Staphylococcus aureus (MRSA) strains could be identified in the milk of dairy cattle in a Paso del Norte region dairy of the United States. Using physiological and PCR-based identification schemes, a total of 40 Staph. aureus strains were isolated from 29 raw milk samples of 133 total samples analyzed. Pulsed-field gel electrophoresis after digestion with the SmaI enzyme revealed that the 40 confirmed strains were represented by 5 pulsed-field types, which each contained 3 or more strains. Of 7 hospital strains isolated from cows undergoing antibiotic therapy, 3 demonstrated resistance to 3 or more antimicrobial classes and displayed similar pulsed-field gel electrophoresis patterns. A secondary purpose of this study was to elucidate the evolutionary relationships of strains isolated in this study to genomically characterized Staph. aureus strains. Therefore, Roche 454 GS (Roche Diagnostics Corp., Dallas, TX) pyrosequencing was used to produce draft genome sequences of an MRSA raw milk isolate (H29) and a methicillin-susceptible Staph. aureus (PB32). Analysis using the BLASTn database (http://blast.ncbi.nlm.nih.gov/) demonstrated that the H29 draft genome was highly homologous to the human MRSA strain JH1, yet the ß-lactamase plasmid carried by H29 was different from that carried by JH1. Genomic analysis of H29 also clearly explained the multidrug resistance phenotype of this raw milk isolate. Analysis of the PB32 draft genome (using BLASTn) demonstrated that this raw milk isolate was most related to human MRSA strain 04-02981. Although PB32 is not a MRSA, the PB32 draft genome did reveal the presence of a unique staphylococcal cassette mec (SCCmec) remnant. In addition, the PB32 draft genome revealed the presence of a novel bovine staphylococcal pathogenicity island, SaPIbovPB32. This study demonstrates the presence of clones closely related to human and (or) bovine Staph. aureus strains circulating in a dairy herd.

A search for Drosophila neural precursor genes identifies ran.

The Drosophila ran gene has been isolated in a differential cDNA screen designed to identify genes that are dynamically expressed in embryonic neuroblasts. The guanine triphosphate (GTP)-binding Ran protein, a member of the Ras superfamily, has been shown to participate in a variety of transport related processes in other organisms. Drosophila ran codes for a 216 amino acid (aa) protein that shares 78% and 86% identity with the yeast and human Ran proteins, respectively. Database searches have identified a second Drosophila ran gene, ran-like. The predicted Ran-like protein shares 59% identity with its isoform. Embryo in situ mRNA localization of ran and ran-like expression reveals that both are maternally expressed; however zygotic ran expression is restricted to central nervous system (CNS) neuroblasts undergoing late lineage formation, while ran-like expression is detected in the developing trachea and salivary gland. To investigate the significance of ran-restricted CNS expression, we have targeted its misexpression to different temporal windows of CNS development. In addition, a dominant-negative mutant form of ran was targeted to the developing CNS and to the larval eye/antenna imaginal disc to assess the role of ran-dependent functions. Embryonic CNS misexpression of the mutant, but not wild-type, ran results in larval death. Neither wild-type nor mutant ran misexpression had any detectable effect on embryonic CNS lineage specification, nuclear transport of a number of CNS-specific transcription factors or axonal guidance. However, expression of the dominant-negative mutant ran in the developing eye/antenna disc did result in a severe adult eye phenotype marked by apoptosis of photoreceptor, cone and pigment cells.

The IA-2 gene family: homologs in Caenorhabditis elegans, Drosophila and zebrafish.

AIMS/HYPOTHESIS: IA-2 and IA-2beta are major autoantigens in Type I (insulin-dependent) diabetes mellitus and are expressed in neuroendocrine tissues including the brain and pancreatic islets of Langerhans. Based on sequence analysis, IA-2 and IA-2beta are transmembrane protein tyrosine phosphatases but lack phosphatase activity because of critical amino acid substitutions in the catalytic domain. We studied the evolutionary conservation of IA-2 and IA-2beta genes and searched for homologs in non-mammalian vertebrates and invertebrates. METHODS: IA-2 from various species was identified from EST sequences or cloned from cDNA libraries or both. Expression in tissues was determined by transfection and in situ hybridization. RESULTS: We identified homologs of IA-2 in C. elegans, Drosophila, and zebrafish which showed 46, 58 and 82 % identity and 60, 65 and 87 % similarity, respectively, to the amino acids of the intracellular domain of human IA-2. Further studies showed that IA-2 was expressed in the neural tissues of the three species. Comparison of the genomic structure of the intracellular domain of human IA-2 with that of human IA-2beta showed that they were nearly identical and comparison of the intron-exon boundaries of Drosophila IA-2 with human IA-2 and IA-2beta showed a high degree of relatedness. CONCLUSION/INTERPRETATION: Based on these findings and sequence analysis of IA-2 homologs in mammals, we conclude that there is an IA-2 gene family which is a part of the larger protein tyrosine phosphatase superfamily. The IA-2 and IA-2beta genes represent two distinct subgroups within the IA-2 family which originated over 500 million years ago, long before the development of the pancreatic islets of Langerhans.

Nerfin-1 and -2, novel Drosophila Zn-finger transcription factor genes expressed in the developing nervous system.

To gain insight into the regulatory networks controlling Drosophila neural-identity decisions, we have identified new neuronal precursor genes by performing an in situ hybridization screen of differentially selected embryonic head cDNAs. Here, we describe the molecular characteristics and expression profile of nerfin-1, a novel pan-neural precursor gene. This paper also documents the embryonic expression of another structurally related gene, nerfin-2. During early CNS development, nerfin-1 gene expression is activated in neuroblasts (NBs) prior to lineage formation. However, after early sublineage development, nerfin-1 expression shifts from NBs to ganglion mother cells (GMCs) but is not expressed in neurons or glia. Differing from nerfin-1, nerfin-2 is expressed only in a subset of brain neurons. Possessing a conserved putative DNA-binding domain, the predicted Nerfin-1 and -2 proteins belong to a subfamily of Zn-finger transcription factors with cognates identified in nematode, mouse and man.

Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development.

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas (R. Kambadur et al., 1998, Genes Dev. 12, 246-260). Seeking to identify the cellular mechanisms that generate these expression compartments, we studied the lineage development of isolated NBs in culture. We found that the Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. Our results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions generating additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb --> Pdm --> Cas --> Gh expression during NB outgrowth.

Compromised HOXA5 function can limit p53 expression in human breast tumours.

Expression of the p53 gene protects cells against malignant transformation. Whereas control of p53 degradation has been a subject of intense scrutiny, little is known about the factors that regulate p53 synthesis. Here we show that p53 messenger RNA levels are low in a large proportion of breast tumours. Seeking potential regulators of p53 transcription, we found consensus HOX binding sites in the p53 promoterS. Transient transfection of Hox/HOXA5 activated the p53 promoter. Expression of HOXA5 in epithelial cancer cells expressing wild-type p53, but not in isogenic variants lacking the p53 gene, led to apoptotic cell death. Moreover, breast cancer cell lines and patient tumours display a coordinate loss of p53 and HOXA5 mRNA and protein expression. The HOXA5 promoter region was methylated in 16 out of 20 p53-negative breast tumour specimens. We conclude that loss of expression of p53 in human breast cancer may be primarily due to lack of expression of HOXA5.

Neuroblast pattern formation: regulatory DNA that confers the vnd/NK-2 homeobox gene pattern on a reporter gene in transgenic lines of Drosophila.

DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the upstream regulatory region of the vnd/NK-2 gene were cloned in the 5'-flanking region of a beta-galactosidase (beta-gal) reporter gene in the P-element pCaSpeR-AUG-beta-gal, and the effects of the DNA on the pattern and time of expression of beta-gal were determined in transgenic embryos. Embryos from 11 lines transformed with -8.4 kb of vnd/NK-2 regulatory DNA expressed beta-gal patterns that closely resemble those of vnd/NK-2. In embryos from four lines transformed with -5.3 kb of vnd/NK-2 DNA, beta-gal was found in the normal vnd/NK-2 pattern in the nerve cord but not in part of the cephalic region. beta-Gal patterns in embryos from transgenic lines containing -0.57, -2.2, or -2.9 kb of vnd/NK-2 DNA did not resemble vnd/NK-2. Null vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to +0.34 beta-gal] expressed little beta-gal in contrast to siblings with a wild-type vnd/NK-2 gene. We conclude that (i) the 8.4-kb DNA fragment from the vnd/NK-2 gene contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, sequences that may be involved in the switch between neuroblast vs. epidermoblast pathways of development, (ii) the 5'-flanking region of the vnd/NK-2 gene between -5.3 and -8. 4 kb is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region, and (iii) vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression.

Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS.

POU transcription factors participate in cell-identity decisions during nervous system development, yet little is known about the regulatory networks controlling their expression. We report all known Drosophila POU genes require castor (cas) for correct CNS expression. drifter and I-POU depend on cas for full expression, whereas pdm-1 and pdm-2 are negatively regulated. cas encodes a zinc finger protein that shares DNA-binding specificity with another pdm repressor: the gap segmentation gene regulator Hunchback (Hb). Our studies reveal that the embryonic CNS contains sequentially generated neuroblast sublineages that can be distinguished by their expression of either Hb, Pdm-1, or Cas. Hb and Cas may directly silence pdm expression in early and late developing sublineages, given that pdm-1 cis-regulatory DNA contains >=32 Hb/Cas-binding sites and its enhancer(s) are ectopically activated in cas- neuroblasts. In addition, the targeted misexpression of Cas in all neuroblast lineages reduces Pdm-1 expression without altering Hb expression. By ensuring correct POU gene expression boundaries, hb and cas maintain temporal subdivisions in the cell-identity circuitry controlling CNS development.

Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila. A putative lipophorin.

A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. Here we provide the first molecular description of the Drosophila gene encoding a retinoid- and fatty acid-binding glycoprotein (RFABG) and present evidence that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the approximately 70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the approximately 70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects.

Pollux, a novel Drosophila adhesion molecule, belongs to a family of proteins expressed in plants, yeast, nematodes, and man.

Adhesion molecules have pivotal roles in cellular processes critical to the development and maintenance of multicellular organisms. Here we describe a new member of the adhesive repertoire encoded by the Drosophila pollux (plx) gene. Marked by a novel 74-amino-acid domain, Plx belongs to a highly conserved family with members in plants, yeast, nematodes, and man, including the human oncoprotein TRE17. Essential for viability, plx mutant analysis indicates that larval death is attributable to asphyxiation brought on by fluid-congested tracheal tubes. Ultrastructural examination of mutant tracheae reveals defects in cell-extracellular matrix contacts. During embryogenesis, Plx uniformly covers the apical surface of cellular blastoderm cells. It is later found regionally concentrated along subsets of central nervous system axon pathways and on the apical surface of the trachea's tubular epithelium. Cell attachment assays demonstrate that Plx can serve as a ligand for cell surface integrins. Plx also contains a motor neuron-selective adhesive site, multiple proteoglycan-binding motifs, and a leucine zipper: all suggest possible associations with additional components of the adhesion complex.

Misexpression of the white (w) gene triggers male-male courtship in Drosophila.

We report here that the general ectopic expression of a tryptophan/guanine transmembrane transporter gene, white (w), induces male-male courtship in Drosophila. Activation of a hsp-70/miniwhite (mini-w) transgene in mature males results in a marked change in their sexual behavior such that they begin to vigorously court other mature males. In transformant populations containing equal numbers of both sexes, most males participate, thus forming male-male courtship chains, circles, and lariats. Mutations that ablate the w transgene function also abolish this inducible behavior. Female sexual behavior does not appear to be altered by ectopic w expression. By contrast, when exposed to an active homosexual courtship environment, non-transformant males alter their behavior and actively participate in the male-male chaining. These findings demonstrate that, in Drosophila, both genetic and environmental factors play a role in male sexual behavior.

Herpes simplex virus pathogenesis in transgenic mice is altered by the homeodomain protein Hox 1.3.

The DNA sequence TAAT is the core binding motif for the mouse homeodomain protein Hox 1.3 (proposed new name, Hoxa-5). These sequences are present within the multiple TAATGARAT regulatory motifs in the promoters of the immediate-early genes which control herpes simplex virus type 1 replication. To investigate the role of this homeodomain protein in the regulation of herpes simplex virus gene expression and pathogenesis, transgenic mice containing a mouse Hox 1.3 cDNA under the control of the virus- and interferon-inducible Mx 1 promoter were generated. After infection of transgenic mice with herpes simplex virus, Hox 1.3 RNA and protein were expressed at the sites of virus replication. In these transgenic mice, herpes simplex virus replication, spread of virus through the host, and virus-induced mortality were markedly enhanced. Increased spread and replication of herpes simplex virus were also observed in cultured fibroblasts from transgenic mice. This finding suggests that in vivo, Hox 1.3 may increase viral spread by increasing viral replication at the level of the individual infected cells. These results demonstrate that expression of a transgene encoding a single host protein, Hox 1.3, alters the pathogenesis of experimental herpes simplex virus infection. We conclude that a protein that belongs to a class of DNA-binding proteins which are best known for their role in regulating embryonic development may also regulate herpesvirus pathogenesis.

castor encodes a novel zinc finger protein required for the development of a subset of CNS neurons in Drosophila.

Using an enhancer detection screen, we have identified castor, a new gene required for embryonic CNS development in Drosophila. Embryos that lack castor expression have a diminished CNS axonal network and express engrailed aberrantly late in CNS development. castor is unique among the previously described genes involved in Drosophila neurogenesis in that its expression is restricted to a subset of delaminated CNS neuroblasts and to ventral midline glial precursor cells. The putative castor gene product contains a novel zinc-binding domain and multiple transcriptional activation domains, suggesting that it acts as a transcription factor necessary for the development of a subset of CNS neuronal precursors.

Altering the insertional specificity of a Drosophila transposable element.

Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.

The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes.

Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene (Boulay et al., 1987). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) (Sargent and Bennett, 1990). We have named the Drosophila snail-related gene escargot (esg), and the region of sequence conservation common to all three genes the 'snailbox'. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues, escargot is allelic with l(2)35Ce, an essential gene located near snail in the genome.

Progressive restriction in the distribution of the Hox-1.3 homeodomain protein during embryogenesis.

Expression of the murine homeobox containing gene Hox-1.3 was analyzed in mouse embryos using polyclonal antisera to peptides predicted from cDNA and genomic sequences. At the earliest stage examined, 7.5 days gestation, cell nuclei throughout the three embryonic germ layers and in extraembryonic structures derived from the fertilized ovum were strongly immunoreactive. Rostro-caudal gradients or other patterns of regional differentiation in levels of expression could not be seen. Surrounding maternal tissue showed only weak immunoreactivity. At 8.5 days gestation, immunoreactivity was present in all embryonic structures including neural tube, somites and lateral plate mesoderm, ectoderm and endoderm. Immunoreactivity was progressively restricted thereafter. At 17 days gestation, strong immunoreactivity was largely restricted to the nervous system, both central and peripheral. Spinal cord was well stained, with a dramatic reduction in intensity near the junction of spinal cord and brain. In addition to this overall pattern, enhanced immunoreactivity appeared in limited populations of newly-formed neuroblasts of spinal cord and brain, suggesting that Hox-1.3 might serve to regulate the development of specific types of neurons following cessation of precursor cell mitosis.

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase.

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.

Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1.3 genes.

A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

The Hox-1.3 homeo box protein is a sequence-specific DNA-binding phosphoprotein.

We report that the murine Hox-1.3 homeo domain protein is a nuclear phosphoprotein capable of binding to specific DNA sequences. DNase I protection of the Hox-1.3 gene promoter region with the Hox-1.3 protein identifies a binding site 144 bp upstream from the start of transcription. Both phosphorylated and nonphosphorylated forms bind DNA directly in a sequence-specific manner. Electrophoretic mobility shift assays were performed with a set of synthetic oligonucleotides representing either the DNase I-protected region of the Hox-1.3 gene or partially homologous sequences present in promoter regions of other characterized viral, yeast, and mammalian genes. From the results, we deduce a consensus binding motif of CPyPyNATTAT/GPy. Base substitutions in the core ATTA sequence severely reduce or abolish binding. In the SV40 enhancer, the Hox-1.3 binding motif overlaps both the octamer (Octa2) and the transactivator protein-1 (AP-1) binding sites. The Hox-1.3 binding motif also overlaps the nuclear factor III (NF-III) octamer motif in the adenovirus-2 origin of DNA replication. Overlap among DNA-binding sites suggests that regulation imparted by certain cis-elements may be integrated by these different factors.

Differential expression of the homeobox gene Hox-1.3 in F9 embryonal carcinoma cells.

The Hox-1.3 gene is located on mouse chromosome 6 and has been previously shown to be expressed in mouse embryos and adults. In this study, we have examined the steady-state levels of the Hox-1.3 transcripts in undifferentiated and differentiated F9 embryonal carcinoma cells. We find that there is a rapid increase of Hox-1.3 transcripts after differentiation induction of F9 cells. The level of the major 1.85-kilobase (kb) transcript peaks at 16-24 hr after differentiation induction of F9 cells. By using primer extension techniques the 5' ends of the major 1.85-kb transcript have been mapped to two sites in induced F9 cells. Cellular fractionation of RNA and transfer blot gel analysis has localized one minor transcript to the nucleus, whereas the major transcript and two additional minor transcripts appear in the nucleus and the cytoplasm of induced F9 cells. The results of nuclear run-off experiments with uninduced and induced F9 cell nuclei indicate that there is a substantial increase in the rate of Hox-1.3 transcription upon induction of F9 cells with retinoic acid.