Activation of the Lck tyrosine protein kinase by the Herpesvirus saimiri tip protein involves two binding interactions.

The Tip protein of Herpesvirus saimiri strain 484C binds to and activates the Lck tyrosine protein kinase. Two sequences in the Tip protein were previously shown to be involved in binding to Lck. A proline-rich region, residues 132-141, binds to the SH3 domain of the Lck protein. We show here that the other Lck-binding domain, residues 104-113, binds to the carboxyl-terminal half of Lck and that this binding does not require the Lck SH3 domain. Mutated Tip containing only one functional Lck-binding domain can bind stably to Lck, although not as strongly as wild-type Tip. Interaction of Tip with Lck through either Lck-binding domain increases the activity of Lck in vivo. Simultaneous binding of both domains is required for maximal activation of Lck. The transient expression of Tip in T cells was found to stimulate both Stat3-dependent and NF-AT-dependent transcription. Mutant forms of Tip lacking one or the other of the two Lck-binding domains retained the ability to stimulate Stat3-dependent transcription. Tip lacking the proline-rich Lck-binding domain exhibited almost wild-type activity in this assay. In contrast, ablation of either Lck-binding domain abolished the ability of Tip to stimulate NF-AT-dependent transcription. Full biological activity of Tip, therefore, appears to require both Lck-binding domains.

Direct binding and activation of STAT transcription factors by the herpesvirus saimiri protein tip.

The Tip protein from Herpesvirus saimiri specifically binds to and activates the protein tyrosine kinase, p56(lck). It has been demonstrated that the expression of Tip in T cells is capable of inducing the DNA binding of members of the signal transducers and activators of transcription (STAT) family of transcription factors. We have examined the mechanism behind which STATs 1 and 3 are activated by Tip expression. Tip becomes tyrosine phosphorylated by p56(lck) at two sites in the amino-terminal tail region. One site of phosphorylation lies within a consensus YXPQ binding motif for the SH2 domains of STATs 1 and 3. We demonstrate that tyrosine phosphorylation of Tip at this site is required for the binding of STATs, and the induction of STAT dependent transcription. Furthermore, we demonstrate that, similar to STAT activation by v-Src, the optimum induction of STAT-dependent transcription by Tip requires Ras/Rac mediated signaling events.

Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.

Inhibition of cell fate in Drosophila by Enhancer of split genes.

The neurogenic genes of Drosophila act during many different times and places during development. It is thought their role is to repress cell fate within a group of equivalent cells and thus allow the singling out of discrete numbers of precursors. Amongst the genes at the neurogenic locus, Enhancer of split is a family of seven related genes that encode proteins containing the basic helix-loop-helix motif characteristic of transcriptional regulators. Previous functional analyses of these genes have relied on deletions which eliminate many other genes. We have ectopically expressed two of the Enhancer of split basic helix-loop-helix genes, m5 and m8, to test their effect on the determination of the precursor cells of adult sensory organs. Ectopic expression of m5 or m8 before bristle precursor division results in loss of sensory bristles from all parts of the adult fly. Ectopic expression after bristle precursor division produces bristles with aberrant cuticular structures. We have also tested the effect of reducing Enhancer of split gene function using mitotic recombination and show that this de-represses the neural fate and produces supernumerary sensory bristle neurons. We conclude that the Enhancer of split basic helix-loop-helix genes inhibit neural fate during the selection of neural precursors, and that they also play a role in restricting the neuronal fate to one of the four progeny cells of the bristle precursor.

The use of new technologies by rural family physicians.

BACKGROUND: Although office procedures that involve special training and office equipment are often performed by a specialist in an urban setting, they are increasingly being performed by family physicians in rural settings. This study documents the prevalence of four such procedures in rural family practice: flexible sigmoidoscopy, cardiac stress testing, colposcopy, and nasopharyngoscopy. Individual and community characteristics of physicians who perform each of the procedures are compared with those of physicians who do not. METHODS: Data were collected on office technology and the characteristics of physicians, their practices, and their communities through telephone interviews with 403 randomly selected, rural family physicians and general practitioners in eight states. Descriptive and univariate analyses were used. RESULTS: Flexible fiberoptic sigmoidoscopy was performed by 57% of the physicians in our sample. The presence of another physician in the group or in the community who performed this procedure increased the probability of a rural physician performing it. Being male, recent licensure, board certification, and patient volume were also positively associated with the performance of this procedure. CONCLUSIONS: This study found evidence of a collegial effect among rural physicians and of a significant number of rural physicians seeking postresidency training in new procedures.

The role of the enhancer of split complex during cell fate determination in Drosophila.

Molecular and genetic data predict that the Enhancer of split locus functions at the end of a pathway dictating appropriate cell fate determination in a number of developmental contexts. We have sought to dissect the role individual member genes of the complex play through a molecular analysis. Of the two principle class of genes, the first, members of the basic helix-loop-helix (bHLH) class of proteins are expressed in specific regions of the embryo in subtle, overlapping patterns in cells that will differentiate as epidermis. The second, groucho, a member of the WD40 class of proteins, is expressed more generally. Immunoprecipitation experiments do not implicate groucho in G protein mediated signal transduction, a known function of many WD40 type proteins. Instead, the nuclear localisation of the protein suggests a relationship to the bHLH members of the complex. Differences in expression of the bHLH genes between neurogenic mutants implies two pathways to their activation during epidermal determination.

Two genetically and molecularly distinct functions involved in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster.

Molecular correlation of the genetic aspects of the function of the neurogenic gene Enhancer of split [E(spl)] has previously been hampered by the densely transcribed nature of the chromosomal region within which it resides. We present data indicating that two distinct molecular species contribute to E(spl) function. Analysis of new E(spl) alleles has allowed us to define two complementing functions within the locus. Subsequent phenotypic analysis of different E(spl) deficiencies combined with P element-transformed constructs has demonstrated that these two functions correspond to: (1) a family of helix-loop-helix (HLH) protein-encoding genes and (2) the single copy gene E(spl) m9/10, whose product shares homology with G-protein beta subunits. The zygotically active E(spl) HLH genes can, at least partially, substitute for one another's functions and their total copy number determines the activity of the locus. E(spl) m9/10 acts synergistically with the E(spl) HLH genes and other neurogenic genes in the process of neurogenesis. The maternal component of E(spl) m9/10 has the most pronounced effect in neurogenesis, while its zygotic component is predominantly required during postembryonic development. The lethality of trans-heterozygotes of null E(spl) deficiency alleles with a strong Delta point mutation is a result of the concomitant reduction in activity of both E(spl) HLH and m9/10 functions. Immunocytochemical localization of the E(spl) m9/10 protein has revealed that it is a ubiquitously distributed nuclear component in embryonic, larval and imaginal tissues.

Early neurogenesis.

After the symmetry of the early embryo is disrupted by morphogenetic movements, one of the first differentiation events is the appearance of discrete neural precursors, the neuroblasts, which segregate from a defined region of ectoderm. Within this region, presumptive epidermal precursors are intermingled with the neuroblasts. Sorting between these fates appears to depend upon cell-cell interactions and requires the function of two sets of genes; one whose mutant phenotype results in an excessive central nervous system (CNS) and the other a subnormal CNS. Molecular cloning of these genes reveals striking homologies which can place the products into two camps. The first are recognizable cell surface components (and potential signal transducers), and the second DNA binding proteins, plausibly transcription factors.

A deduced gene product from the Drosophila neurogenic locus, enhancer of split, shows homology to mammalian G-protein beta subunit.

The correct segregation of neural from epidermal lineages in Drosophila embryogenesis depends on the activity of the six zygotic "neurogenic" genes. One of the neurogenic genes, Enhancer of split, is particularly noteworthy in its genetic interactions with Notch and Delta, which both appear to code for transmembrane proteins with homology to the epidermal growth factor. Transformation experiments have demonstrated the cloning of sequences necessary for Enhancer of split gene function. We report here that the gene product derived from DNA sequencing shows homology to the beta subunit of mammalian G proteins and CDC4, a yeast cell cycle gene. We demonstrate that expression of the transcripts relates to the developing central nervous system. These data suggest a mechanism of interaction between the gene products of Notch and Enhancer of split.

slit: an EGF-homologous locus of D. melanogaster involved in the development of the embryonic central nervous system.

A family of loci homologous to the EGF-like portion of Notch, a gene involved in neurogenesis, have been identified in D. melanogaster. The sequence, spatial, and temporal distribution of both RNA and protein of one of these loci suggest a possible role in the development of the central nervous system (CNS). In situ hybridization and antibody staining of embryos show initial localization in cells along the midline of the neuroepithelium. High level expression is restricted in the developing embryo to a subset of six midline glial cells abutting growing axons. Extracellular localization is suggested by the presence of EGF-like repeats in the deduced protein sequence and antibody staining. Cytological, immunocytochemical, genetic, and molecular data show that this gene corresponds to the slit locus. Mutations in this locus result in the collapse of the regular scaffold of commissural and longitudinal axon tracts in the embryonic central nervous system.

The molecular genetics of Enhancer of split, a gene required for embryonic neural development in Drosophila.

In Drosophila, the very first steps in neurogenesis appear to be controlled by a small group of zygotically acting genes termed the neurogenic loci. Mutations in any of these genes result in a misrouting of epidermal lineages into the neural pathway. Morphological and molecular studies suggest that the correct ectodermal differentiation is mediated by a cell-cell interaction mechanism and that at least some of the neurogenic loci are involved in this mechanism. The molecular analyses of the neurogenic loci Notch and Delta revealed that the putative gene products are large transmembrane proteins with homology to mammalian epidermal growth factor. We describe here a molecular analysis of Enhancer of split [E(spl)], a third neurogenic locus, which displays striking genetic interactions with both Notch and Delta, suggesting a close functional relationship of the respective gene products. We provide evidence for a single genetic complementation group corresponding to a single transcription unit which is necessary for wild-type E(spl) function. P-element-mediated transformation indicates that this transcription unit includes functions associated with both the dominant E(spl)D mutation and the recessive visible allele groucho, and is necessary for the correct differentiation of the embryonic nervous system.

The embryonic expression of the Notch locus of Drosophila melanogaster and the implications of point mutations in the extracellular EGF-like domain of the predicted protein.

The Notch locus of Drosophila melanogaster is one of a small number of zygotically acting 'neurogenic' genes necessary for the correct segregation of neural from epidermal lineages during embryogenesis. The predicted gene product is implicated in a cell interaction mechanism required to achieve this ectodermal differentiation. We have examined wild-type Notch expression by in situ hybridization and find it to be expressed in more cells than we would have predicted given a sole function in regulating neurogenesis. We conclude from these data that Notch plays a more general role in development. In order to assess the dependence of Notch expression on other neurogenic gene function we have hybridized Notch probes to Enhancer of split mutants which are known to interfere with expression of Notch phenotypes. We intimate that the nature of interaction between these genes is not at the level of transcription. Instead, the DNA sequence of split, which is a missense mutation in the EGF-like extracellular domain of the Notch protein, suggests a direct biochemical interaction between Notch and E(spl) proteins. The similar site of a second point mutation, AxE2, implies that protein interactions also occur between Notch proteins. Finally we discuss the general implications of our findings with a view to the models and mechanisms of Notch action in regulating individual cellular interactions during development.

A cytological map of the human X chromosome--evidence for non-random recombination.

The cytological location of six cloned DNA sequences on the human X chromosome has been determined to a high resolution by direct hybridisation 'in situ' to metaphase chromosomes. Each locus has been identified using clones which also detect restriction fragment length polymorphisms by Southern hybridisation. The six loci identified are spaced along the chromosome from Xp22 to Xq28. By combining data obtained using this powerful sequence localisation technique with that from hybrid cell panels and from family studies, it is possible to compare physical and genetic distances, and to demonstrate that the frequency of reciprocal genetic exchange is not uniform along the chromosome length.

Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: location of the gsr (tgs) and iex (crr) genes by specialized transduction.

A lysogen of Escherichia coli K12 with lambda cI857 S7 xis6 nin5 b515 b519 integrated into ptsI was induced and the lysates plated on a Pel- host [on which lambda strains with less than the wild-type amount of DNA form plaques at low frequency (Cameron et al., 1977)]. All of the 40 plaques examined contained phage able to transduce at least two of the genes known from bacteriophage P1 transduction experiments to be closely linked to ptsI. Assuming that each specialized transducing phage arose by a single illegitimate recombination event, the distribution of phage types showed that the gene order is cysA gsr ptsI (ptsH, iex) cysZ lig; both gsr+ and iex+ were dominant. Analysis of restriction endonuclease digests of the transducing phage confirmed that no unexpected DNA rearrangements had taken place and allowed the construction of a map of the sites of action of the restriction endonucleases EcoRI, HindIII, BamI and Kpn for over 20 kilobases of E. coli DNA. In an Appendix, we show cysA and cysZ mutants to be deficient in sulphate assimilation.