Biomedical subjects

J J Bonner

Publications and source records attributed to J J Bonner.

At least 19 recordsLinked to original sources

PubMed1995

Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast.

The GATA motif (WGATAR) is found in the promoter regions of numerous Caenorhabditis elegans genes, including two intestine-specific genes, vit-2 and ges-1, in which it has been shown to be required for promoter function. The protein ELT-1, encoded by a single-copy gene homologous to the GATA family of vertebrate transcription factors, is potentially capable of interacting with this element. In order to determine whether ELT-1 is a transcriptional activator that recognizes this sequence, we have expressed it under the control of the GAL1 promoter in yeast. lacZ driven by the CYC1 promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. beta-Galactosidase was expressed upon induction only when GATA sequences were present, and expression was increased dramatically by additional binding sites. Deletion analysis demonstrated that the C terminus, containing only one of the two zinc fingers, is sufficient for activation. In addition, the DNA-binding domain and two transactivation regions were identified by fusing these isolated domains to previously defined domains of heterologous transcription factors. While most single base alterations in the GATA core sequence eliminated activity, an A to C change in position four, creating a GATC core, was found to increase activity significantly. The deleted ELT-1 protein containing only the C-terminal Zn finger was sufficient for activation in response to GATA, but both fingers were required for activation at GATC. A variety of sites with non-optimal sequences surrounding the GATA core also were found to be excluded better by the protein containing both Zn fingers. Furthermore, a fusion protein containing the entire ELT-1 DNA binding domain fused to the VP16 activation domain was found to have an even greater preference for the GATC core, as well as the optimal flanking bases. We conclude that, although ELT-1 having only its C-terminal finger is capable of activation in response to the WGATAR site, the presence of the upstream finger supplies additional base specificity.

Amino Acid Sequence↗

PubMed1995

Genetic identification of the site of DNA contact in the yeast heat shock transcription factor.

The heat shock transcription factor (HSF), a trimeric transcription factor, activates the expression of heat shock genes in eukaryotes. We have isolated mutations in the HSF1 gene from Saccharomyces cerevisiae that severely compromise the ability of HSF to bind to its normal binding site, repeats of the module nGAAn. One of these mutations, Q229R, shows a "new specificity" phenotype, in which the protein prefers the mutant sequence nGACn. These results identify the region of HSF that contacts DNA, in complete agreement with the crystal structure of HSF of Kluyveromyces lactis and the nuclear magnetic resonance data from HSF of Drosophila melanogaster. To determine the orientation of the DNA-binding domain on the nGAAn motif, we performed site-specific cross-linking between cysteine residues of single-cysteine substitutions. Cysteines placed at the N terminus of the DNA contact helix formed cross-links readily, while cysteines placed at the C terminus of the helix did not.

Base Sequence↗

PubMed1994

Interactions between DNA-bound trimers of the yeast heat shock factor.

The heat shock transcription factor (HSF) is a trimer that binds to DNA containing inverted repeats of the sequence nGAAn. HSF can bind DNA with the sequence nGAAnnTTCn or with the sequence nTTCnnGAAn, with little preference for either sequence over the other. However, (nGAAnnTTCn)2 is considerably less active as a heat shock response element (HSE) than is (nTTCnnGAAn)2. The electrophoretic mobilities of DNA-protein complexes and chemical cross-linking between protein monomers indicate that the sequence (nGAAnnTTCn)2 is capable of binding a single HSF trimer. In contrast, the sequence with higher biological activity, (nTTCnnGAAn)2, is capable of binding two trimers. Thus, the ability of four-nGAAn-element HSEs to bind one or two trimers depends on the permutation with which the elements are presented. A survey of naturally occurring HSEs shows the sequence (nTTCnnGAAn)2 to be the more prevalent. We suggest that the greater ability of one permutation over the other to bind two HSF trimers accounts for the initial identification of the naturally occurring heat shock consensus sequence as a region of dyad symmetry.

Base Sequence↗

PubMed1992

Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae.

The HSF1 gene of Saccharomyces cerevisiae directs the synthesis of the heat shock transcription factor, HSF. The gene is essential; disruption mutations are lethal. Using a plasmid shuffle screen, we isolated mutations in the HSF1 gene after in vitro mutagenesis of plasmid DNA with hydroxylamine. From a collection of both conditional (temperature-sensitive) and unconditional lethal mutations, we recovered mutations that map exclusively to the 5' half of the gene. All are nonsense mutations, including conditional mutations that map 5' to the portion of the HSF1 gene that encodes the DNA-binding domain of the transcription factor. For one such mutation, we demonstrated that the nonsense mutation is subject to translational readthrough, even though there are no known nonsense suppressors in the genetic background of our strain. Our results suggest that the HSF protein is highly tolerant of amino acid changes, a conclusion that is consistent with the very low degree of evolutionary conservation among HSF proteins. Our results also suggest that translational readthrough occurs with moderate efficiency in yeast, particularly when the terminator codon is followed immediately by an A or C residue. This result illustrates that the inference of gene function from mutant phenotype depends critically upon the analysis of a true null allele, and not merely an amber or ochre allele.

Amino Acid Sequence↗

PubMed1992

Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor.

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.

Base Sequence↗

PubMed1991

Vectors for the expression and analysis of DNA-binding proteins in yeast.

A series of 13 vectors is described. All are yeast centromere plasmids with the LEU2 gene for selection in yeast, and pUC19 sequences for growth in Escherichia coli. All contain the GAL1 promoter directing transcription into a multiple cloning site (MCS). For twelve of the plasmids, synthetic oligodeoxyribonucleotides create an ATG start codon, in a productive context for yeast, prior to the MCS. Spacing between the ATG and the MCS is variable, to facilitate the cloning of gene fragments in the appropriate reading frame. Nine of the plasmids also contain the strong transcriptional activator from the herpes simplex virus VP16 gene, joined downstream from the MCS. In these nine vectors, all possible combinations of reading frames are available. The suitability of these plasmids for the expression and analysis of DNA-binding domains is tested by cloning into them fragments of the yeast HSF1 gene, encoding the heat shock transcription factor (HSF). The regulation of reporter gene expression by the chimeric HSF-VP16 fusions is described, as is the utility of these vectors for other applications.

Base Sequence↗

PubMed1991

Genetic analysis of chromosome region 63 of Drosophila melanogaster.

The salivary chromosome region including cytological division 63 of Drosophila melanogaster was genetically analyzed in order to (1) characterize this previously unstudied region and (2) attempt to isolate mutations in the hsp82 gene. Seven deletions which span this region were isolated, including four which remove the hsp82 gene. A Minute mutation was mapped to this region and this Minute was used to isolate duplications in the 63 region. These duplications map the Minute to 63B8-C1. F2 screens were initiated using deletions which remove the hsp82 gene. Over 15,000 chromosomes were screened, yielding 40 lethal mutations which comprise 14 complementation groups. Several of these mutations map outside the 63 region and appear to give second site interaction with the Minute locus. Four loci, including the Minute gene, are candidates for hsp82 mutations by cytogenetic mapping. These loci were tested for complementation with a P element carrying the hsp82 gene. However, none of the mutations was rescued.

Animals↗

PubMed1989

Use of promoter fusions in Drosophila genetics: characterization of a YP1-ADH fusion gene.

In Drosophila melanogaster the yolk protein (YP) genes are normally expressed only in the fat body and follicular epithelium of adult females--never in males or in larvae. We describe here a first step toward a genetic examination of the developmental controls that restrict the activity of the YP genes to adult female tissues. A YP1 promoter that contains the tissue-, temporal-, and sex-specific controlling elements for expression was fused to the reporter gene, alcohol dehydrogenase (Adh). The gene fusion was transformed into an Adh-deficient genotype. As assayed by a number of criteria, that the fusion gene is expressed in the same physiological manner as the endogenous yolk protein genes. The fusion gene's activity is modulated in trans by a temperature-sensitive allele of the sex determination gene, tra-2. The Adh enzyme serves as a selectable marker and therefore these flies are suitable for use in genetic screens for trans-acting mutations that affect the expression of the yolk protein genes.

Alcohol Dehydrogenase↗

PubMed1989

Electron microscopical analysis of Drosophila polytene chromosomes. V. Characteristics of structures formed by transposed DNA segments of mobile elements.

An electron microscopical (EM) analysis was performed on regions of polytene chromosomes which contained DNA segments of different genetic composition, inserted by P element-mediated transformation into the Drosophila melanogaster genome. In seven of ten regions examined, containing insertions of the hsp28-ry, hsp70-Adh, ryhsp 70-beta-gal genes and of the ry gene tetramer, new bands appeared. Lack of new bands in three other strains is apparently connected with the fusion of the inserted material to preexisting bands. The new bands do not differ morphologically from the usual bands of polytene chromosomes, and their formation is likely due to predominant insertion of DNA segments into interbands. Among the constructs examined, the minimal length of a DNA segment which appears as a new band is about 5 kb; the DNA packing ratio in the new bands varies from 30 to 50. Activation of the inserted genes by heat shock has enabled us to observe the puffing characteristics of new bands. A sequence of some one kb forms a large interband, or micropuff; the puff size is correlated with the length of the genes being activated. If a DNA segment contains a single gene, then its activation causes the decompaction of the whole band; however, when a DNA segment consists of two genes and the promoter element of the activated gene is positioned in the middle of the sequence, the band splits and only part is decompacted and puffed. The DNA packing ratio in the puffs is 1.4-3.5. The subsequent deletion of the hsp70 promoter but retention of 23, 59, and 73 by from the transcription start points leads to failure of puff formation. In all the transformed sites an increase in the total length of the interbands adjacent to the insert as compared with the initial interband was observed. This increase appears to be due to decompaction of the P element DAN flanking the inserted segments. It is shown that a DNA segment, consisting of four tandemly repeated ry gene copies and interspersed by material which includes P DNA, forms a complex of loose chromatin in which, however, four bands can be resolved. We also observed a lengthening of interband regions containing only the P element sequence itself. Insertion of the complete 2.9 kb P element into the large single 10A1-2 bound of the X chromosome (an insertion in the region approximately 10 kb to the right of the v gene) causes splitting of the band into two parts and formation of a new interband.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Genetic analysis of chromosomal region 67A-D of Drosophila melanogaster.

In an effort to (1) characterize the 67 interval of chromosome 3 of Drosophila melanogaster genetically and (2) isolate mutations of the 67B1 small heat shock protein (hsp) gene cluster specifically, we undertook a mutational analysis of the 67A-D subinterval. Using a deficiency of the 67A2 to 67D11-13 region, Df(3L)AC1, we screened 8700 diepoxybutane-treated chromosomes and 7800 ethyl methanesulfonate-treated chromosomes for visible and lethal mutations throughout this interval and recovered 74 independent recessive lethal mutations, but no visible mutations. One of the lethal mutations, d29A6, was identified as an overlapping deficiency extending from 66F3 to 67B1. An additional 6000 diepoxybutane-treated chromosomes were screened for lethality over d29A6, yielding another four lethal mutations within the 67A2-B1 subinterval. These 78 lethal mutations, along with two others isolated in other laboratories, define 23 essential loci--6 within the 67A2-B1 subinterval and 17 within the 67A2 to D11-13 subinterval. Many of these loci appear to be required for imaginal development only, exhibiting late larval to pharate adult lethal phases. Examination of the 67A2-B1 lethal complementation groups for (1) earlier onset of lethality following a heat shock, (2) missing or altered small hsps on two-dimensional protein gels, and (3) restoration of viability by transformed wild-type copies of the small hsp genes indicates that none of these mutations affect the small hsps. On the basis of this analysis and the known homology of the genes, we conclude that the small hsps are functionally equivalent.

Animals↗

PubMed1987

Mutations that induce the heat shock response of Drosophila.

We have isolated a number of mutations in D. melanogaster that result in the constitutive expression of the heat shock response in a tissue-specific manner. These mutations induce alcohol dehydrogenase (ADH) when the ADH structural gene is fused to the promoter for the 70 kd heat shock protein (hsp70) gene. Flies carrying these mutations, the hsp70-Adh fusion, and a deletion in their endogenous Adh genes are ethanol tolerant and exhibit elevated ADH levels. Several of the tissue-specific mutations have also been shown to induce an hsp26-Adh fusion gene in trans. The mutation Act88FKM75, a G----A transition in the indirect flight muscle-specific actin gene, also exhibits this phenotype. Comparisons with the Act88FKM75 mutation suggest that the tissue-specific mutations induce the heat shock response by disrupting the physiology of the cells in which the variant gene product is expressed.

Alcohol Dehydrogenase↗

PubMed1986

Deletion polymorphism in a Drosophila melanogaster heat shock gene.

We have continued the transcriptional analysis of the region of cytological locus 67B that contains the four small heat shock genes and other genes. Transcription from one of the heat shock genes in the region, hsp 26, takes place during high temperature treatment and at certain developmental stages, without heat shock, in several tissues, such as imaginal discs and adult ovaries. Observations of unexpected products after nuclease protection experiments provided the first indication of what genomic blot experiments showed to be small deletions. The alleles containing the deletion are expressed at the same level as the wild type allele. The deletion shortens the protein product, implying that it is in the coding region. Furthermore, flies homozygous for one of the deletion alleles are viable.

Alleles↗

PubMed1986

Small heat shock proteins of Drosophila associate with the cytoskeleton.

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vimentin-like intermediate filament proteins (IFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mechanism. Indirect immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe--indeed, lethal--shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the same perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.

Animals↗

PubMed1986

Major histocompatibility complex influences reproductive efficiency: evolutionary implications.

Measurements of six reproductive characteristics revealed differences in seven congenic mouse strains with genetic differences in H-2, the major histocompatibility complex (MHC). Associations between reproductive efficiency and MHC has evolutionary implications. One association explains the uneven distribution and heterozygosity of MHC alleles in wild mouse and human populations. The second is based on links between MHC and hormonal sensitivities that regulate an individual's responses to the environment. MHC may be a seat of genotypic-environmental interactions that guide natural selection.

Alleles↗

PubMed1985

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila.

Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215ts (ts), only displays a conditional recessive lethality, while the other, RpII215Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/-tissue in gynandromorphs are morphologically normal. cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215+, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/-cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interactions described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term 'negative heterosis' to describe the aforementioned interactions. We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Mandibular skeletal dysmorphology in micrognathic mice.

The primary manifestations of micrognathia were microglossia, midline fusion of the right and left sides of the mandible, total absence of incisor and molar toothbuds and, in many cases, absence or perhaps premature resorption of Meckel's cartilage. In addition, there was altered osteogenesis as evidenced by disrupted trabecular patterns, as well as an overall dimensional reduction of the mandible both antero-posteriorly and laterally. Strikingly similar results were reported by Johnson (1926), who studied the progeny of x-irradiated mice. How specifically our results correlate with this much earlier work is a matter for further analysis. It seems clear that the critical factor in the development of micrognathia is not so much an abnormal formation of the bony mandible, but a deficiency of tongue development, specifically its intrinsic musculature. Thus, mandibular micrognathia involves not only a dysmorphology of the first branchial arch, but also the mesenchymal cell migration from the occipital somites. Taken together, the picture is one that suggests an underlying cause that may have its inception at a much earlier developmental stage, when ectomesenchymal migration from the region of the neural tube occurs. In any event, we can report confidently that spontaneous micrognathia in prenatal mice is not a simple dimensional reduction of the lower jaw, but a more complex morphological phenomenon.

Animals↗

PubMed1984

Glucocorticoid-induced cleft palate genes in chromosome 17: genetic linkage and mapping analyses.

Genes that influence susceptibility to dexamethasone-induced cleft palate and tentatively designated Dep are linked to the major histocompatibility complex H-2 in chromosome 17 of the mouse. Experiments presented refine the map of genes. The results show two or three Dep loci. The two-locus model maps Dep genes to the class II gene E beta and to the chromosomal region between the S and D genes. The three-locus model maps the Dep genes to the chromosomal regions from the centromere to E beta, from E beta to S, and from D to Pgk-2. Experiments were done by comparing the dexamethasone-induced cleft palate dose response of congenic strains with H-2 haplotypes that are recombinants of H-2a and H-2b. The analysis of genetic linkage between H-2 and Dep was expanded to include reciprocal backcrosses. A maternal factor was found to influence the frequency of dexamethasone-induced cleft palate in the backcross fetuses. The factor's origin is associated with the H-2 haplotype of the outcross mother, so the effect is actually a "grandmother effect" that probably is transmitted horizontally. Finally, the sexes were distributed unevenly between the fetuses with cleft palate in two of the congenic strains. This suggests interaction between the H-2-linked Dep genes and a Dep sex-associated gene that modulates susceptibility to dexamethasone-induced cleft palate.

Alleles↗

PubMed1984

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response.

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.

Alcohol Oxidoreductases↗