Biomedical subjects

H M Chamberlin

Publications and source records attributed to H M Chamberlin.

17 recordsLinked to original sources

PubMed2001

Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly.

Combinatorial association of DNA-binding proteins on composite binding sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein-protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 within the N-terminal beta-hairpin motif of its paired domain. The beta-hairpin also participates in recognition of a subset of Pax-5-binding sites. Thus, Pax-5 incorporates protein-protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Together, our results define specific amino acid requirements for Pax-Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.

Amino Acid Sequence↗

PubMed2001

The adaptable lin-39.

Comparative studies of nematode development provide a powerful framework for investigating the evolution of developmental mechanisms. A recent report also demonstrates how comparative work can inform our understanding of basic developmental signaling pathways. In particular, investigation of the differences in vulva development between Caenorhabditis elegans and Pristionchus pacificus has clarified the molecular relationship between an epidermal growth factor-Ras-MAP kinase signaling pathway and downstream Hox transcription factor activity.

Animals↗

PubMed2001

EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development.

The Pax gene egl-38 plays an important role in the development of several organs in C. elegans. To understand how a Pax transcription factor influences distinct developmental choices in different cells and tissue types, we have characterized a second gene, lin-48. lin-48 functions with egl-38 in the development of one structure, the hindgut, but not in other tissues such as the egg-laying system. We show that lin-48 encodes a C2H2 zinc-finger protein that is similar to the product of the Drosophila gene ovo and is expressed in the hindgut cells that develop abnormally in lin-48 mutants. We present evidence that lin-48 is a target for EGL-38 in hindgut cells. We show that lin-48 requires egl-38 for its expression in the hindgut. Using deletion analysis, we have identified two elements in the lin-48 promoter that are necessary for lin-48 expression. We demonstrate that EGL-38 binds with high affinity to one of these elements. In addition, we have observed genetic interactions between mutations in the lin-48 promoter and specific alleles of egl-38. These experiments demonstrate a functional link between Pax and Ovo transcription factors, and provide a model for how Pax transcription factors can regulate different target genes in different cells.

Alleles↗

PubMed2000

Nematode development: an evolutionary fugue.

Recent studies of vulva development in the nematode Pristionchus pacificus have identified cell interactions that do not appear to occur in Caenorhabditis elegans. The new results underscore the diversity of patterning mechanisms that can produce structures with similar cellular morphology.

Animals↗

PubMed2000

The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans.

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Characterization of seven genes affecting Caenorhabditis elegans hindgut development.

We have identified and characterized 12 mutations in seven genes that affect the development of the Caenorhabditis elegans hindgut. We find that the mutations can disrupt the postembryonic development of the male-specific blast cells within the hindgut, the hindgut morphology in both males and hermaphrodites, and in some cases, the expression of a hindgut marker in hermaphrodite animals. Mutations in several of the genes also affect viability. On the basis of their mutant phenotypes, we propose that the genes fall into four distinct classes: (1) egl-5 is required for regional identity of the tail; (2) sem-4 is required for a variety of ectodermal and mesodermal cell types, including cells in the hindgut; (3) two genes, lin-49 and lin-59, affect development of many cells, including hindgut; and (4) three genes, mab-9, egl-38, and lin-48, are required for patterning fates within the hindgut, making certain hindgut cells different from others. We also describe a new allele of the Pax gene egl-38 that is temperature sensitive and affects the conserved beta-hairpin of the EGL-38 paired domain. Our results suggest that a combination of different factors contribute to normal C. elegans hindgut development.

Aging↗

PubMed1998

Lethal mutations defining 112 complementation groups in a 4.5 Mb sequenced region of Caenorhabditis elegans chromosome III.

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2 Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans.

Animals↗

PubMed1998

Cak1 is required for Kin28 phosphorylation and activation in vivo.

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

Animals↗

PubMed1997

The PAX gene egl-38 mediates developmental patterning in Caenorhabditis elegans.

Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted and one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that the egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. We have cloned egl-38 and shown that it is a member of the PAX family of genes, which encode transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution.

Amino Acid Sequence↗

PubMed1996

Three-dimensional structure of human cyclin H, a positive regulator of the CDK-activating kinase.

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at a specific threonine residue. Vertebrate CAK contains two key components: a kinase subunit with homology to its substrate CDKs and a regulatory subunit with homology to cyclins. We have determined the X-ray crystal structure of the regulatory subunit of CAK, cyclin H, at 2.6 A resolution. Cyclin H contains two alpha-helical core domains with a fold similar to that of cyclin A, a regulatory subunit of CAK substrate CDK2, and of TFIIB, a transcription factor. Outside of the core domains, the N- and C-terminal regions of the three structures are completely different. The conformational differences between cyclin H and A structures may reflect functional differences between the two cyclins.

Amino Acid Sequence↗

PubMed1995

Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase.

We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms.

Amino Acid Sequence↗

PubMed1995

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.

Animals↗

PubMed1995

Mutations in the Caenorhabditis elegans gene vab-3 reveal distinct roles in fate specification and unequal cytokinesis in an asymmetric cell division.

Asymmetric cell divisions in which a precursor cell distributes fate potential unequally between the two daughter cells represent one of the major mechanisms for fate specification during development. Such mechanisms suggest at least two distinct cellular activities: factors that act to establish asymmetry in the precursor cell and factors that are distributed or activated unequally and function to make the daughter cells different from each other. In Caenorhabditis elegans, cytokinesis of the first division of the male-specific postembryonic blast cell B is unequal, and the two daughters adopt different fates. Others have observed that the genes lin-17 and lin-44 are required, respectively, to establish and to orient this asymmetric division. Mutations in lin-17 and lin-44 coordinately disrupt cytokinesis and fate specification. We describe the function of the gene vab-3 in the B cell lineage. Mutations in vab-3 disrupt the fate of the anterior daughter of B, B.a. However, unlike lin-17 and lin-44, mutations in vab-3 can disrupt fate without the corresponding disruption of unequal cytokinesis. Analysis of lin-17;vab-3 double mutants suggests that vab-3 acts after lin-17 for B.a. fate specification. Double mutant analysis has also identified additional functions of lin-17 in the B lineage subsequent to this first division.

Animals↗

PubMed1994

The lin-3/let-23 pathway mediates inductive signalling during male spicule development in Caenorhabditis elegans.

During Caenorhabditis elegans male spicule development, four pairs of precursor cells respond to multiple positional cues and establish a pattern of fates that correlates with relative anterior-posterior cell position. One of the extracellular cues is provided by the F and U cells, which promote anterior fates. We show that the genes in the lin-3/let-23 signalling pathway required for hermaphrodite vulval induction also mediate this F/U signal. Reduction-of-function mutations in lin-3, let-23, sem-5, let-60 or lin-45 disrupt the fate of anterior cells. Likewise, activation of the pathway with ubiquitously produced signal results in posterior cells inappropriately adopting the anterior fates even in the absence of F and U. We have further used this genetic pathway to begin to understand how multiple positional cues are integrated to specify cell fate. We demonstrate that lin-15 acts in spicule development as it does in vulval induction, as a negative regulator of let-23 receptor activity. A second extracellular cue, from Y.p, also acts antagonistically to the lin-3/let-23 pathway. However, this signal is apparently integrated into the lin-3/let-23 pathway at some step after lin-45 raf and is thus functionally distinct from lin-15. We have also investigated the role of lin-12 in forming the anterior/posterior pattern of fates. A lin-12 gain-of-function defect is masked by redundant positional information from F and U.

Animals↗

PubMed1993

Multiple cell interactions are required for fate specification during male spicule development in Caenorhabditis elegans.

The B blast cell divides postembryonically in C. elegans males to produce 47 progeny that include all of the cells of the copulatory spicules. During the early development of the B lineage, the anterior daughter of B, B.a, generates eight cells. These cells migrate to form four pairs of cells that flank the developing cloaca (ventral, dorsal, and two identical lateral pairs). For each pair, the more anterior cell produces a distinct lineage ('anterior fate') from the posterior cell ('posterior fate'). For the ventral and dorsal pairs, either cell can migrate to the anterior position and produce the anterior lineage, and the other cell migrates posterior and produces the posterior lineage (Sulston and Horvitz, 1977, Dev. Biol. 56, 110-156). The migration is variable, although the resultant fate pattern is invariant. In the two lateral pairs, both the migration and fate pattern are invariant. Using a laser microbeam to selectively ablate neighboring cells we have found that the cells of the lateral pair also respond to positional cues. For all four pairs other male-specific blast cells provide extracellular cues. In general, F and U promote anterior fates, Y promotes some posterior fates, and the B.a progeny promote posterior fates. Several of these cues are redundant. By ablating combinations of cells we have deduced how these signals may act in concert to specify the fates of the B.a progeny. We propose that fate specification in these pairs depends on three general classes of extracellular cues: positional cues, modulators of positional cues, and lateral signals. The B lineage thus provides an opportunity to study with single cell resolution the integration of multiple intercellular signals.

Animals↗

PubMed1991

Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification.

Within a 7 kb segment of the mtDNA molecule of the root knot nematode, Meloidogyne javanica, that lacks standard mitochondrial genes, are three sets of strictly tandemly arranged, direct repeat sequences: approximately 36 copies of a 102 ntp sequence that contains a TaqI site; 11 copies of a 63 ntp sequence, and 5 copies of an 8 ntp sequence. The 7 kb repeat-containing segment is bounded by putative tRNAasp and tRNAf-met genes and the arrangement of sequences within this segment is: the tRNAasp gene; a unique 1,528 ntp segment that contains two highly stable hairpin-forming sequences; the 102 ntp repeat set; the 8 ntp repeat set; a unique 1,068 ntp segment; the 63 ntp repeat set; and the tRNAf-met gene. The nucleotide sequences of the 102 ntp copies and the 63 ntp copies have been conserved among the species examined. Data from Southern hybridization experiments indicate that 102 ntp and 63 ntp repeats occur in the mtDNAs of three, two and two races of M.incognita, M.hapla and M.arenaria, respectively. Nucleotide sequences of the M.incognita Race-3 102 ntp repeat were found to be either identical or highly similar to those of the M.javanica 102 ntp repeat. Differences in migration distance and number of 102 ntp repeat-containing bands seen in Southern hybridization autoradiographs of restriction-digested mtDNAs of M.javanica and the different host races of M.incognita, M.hapla and M.arenaria are sufficient to distinguish the different host races of each species.

Animals↗

PubMedDate not supplied

EEG, visually evoked and event related potentials in young abstinent alcoholics.

EEG, visually evoked potentials (VEP), and event related potentials (P300) were recorded from 60 males aged 25-40 (20 abstinent alcoholics, 20 social drinkers, and 20 lifetime nondrinkers). Alcoholics were at least 1 month abstinent, medication free, and neuropsychologically normal. Residual effects of alcohol abuse were not detected in EEG power and cortical coupling analyses, or VEP amplitude, latency, and amplitude/intensity slope measures. Only P300 measures, recorded while subjects were actively engaged in a visual oddball task (20% targets), differentiated alcoholics from nonalcoholics. Alcoholics had reduced N2-P3 amplitude and delayed N2 latencies compared to social drinkers and nondrinkers. Because P300 abnormalities have been reported for individuals with positive family history for alcoholism, it is not clear whether these results reflect familial influence or residual effects of alcohol abuse. Issues relating to age, health and medication status of abstinent alcoholics, and use of social drinkers as comparison groups in studies of brain dysfunction in "recovering" alcoholics are also discussed.

Adult↗