Genetics and Genomics Commons^™

Control Of Developmental Timing In Caenorhabditis Elegans, Victor Ambros

Victor R. Ambros

Studies of the nematode Caenorhabditis elegans have identified genetic and molecular mechanisms controlling temporal patterns of developmental events. Mutations in genes of the C. elegans heterochronic pathway cause altered temporal patterns of larval development, in which cells at certain larval stages execute cell division patterns or differentiation programs normally specific for other stages. The products of the heterochronic genes include transcriptional and translational regulators and two different cases of novel small translational regulatory RNAs. Other genes of the pathway encode evolutionarily conserved proteins, including a homolog of the Drosophila Period circadian timing regulator, and a member of the nuclear receptor …

The Lin-41 Rbcc Gene Acts In The C. Elegans Heterochronic Pathway Between The Let-7 Regulatory Rna And The Lin-29 Transcription Factor, Frank Slack, Michael Basson, Zhongchi Liu, Victor Ambros, H. Horvitz, Gary Ruvkun

Victor R. Ambros

Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 …

Structure And Function Analysis Of Lin-14, A Temporal Regulator Of Postembryonic Developmental Events In Caenorhabditis Elegans, Yang Hong, Rosalind C. Lee, Victor R. Ambros

Victor R. Ambros

During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternative lin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternative lin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows …

The Lin-4 Regulatory Rna Controls Developmental Timing In Caenorhabditis Elegans By Blocking Lin-14 Protein Synthesis After The Initiation Of Translation, Philip Olsen, Victor Ambros

Victor R. Ambros

lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA and that acts to developmentally repress the accumulation of LIN-14 protein. This repression is essential for the proper timing of numerous events of Caenorhabditis elegans larval development. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results indicate that the rate of synthesis of lin-14 mRNA, its state of polyadenylation, its abundance in the cytoplasmic fraction, and its polysomal sedimentation profile do not …

The Timing Oflin-4rna Accumulation Controls The Timing Of Postembryonic Developmental Events Incaenorhabditis Elegans, Rhonda Feinbaum, Victor Ambros

Victor R. Ambros

The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of …

Cell Cycle-Dependent Sequencing Of Cell Fate Decisions In Caenorhabditis Elegans Vulva Precursor Cells, Victor Ambros

Victor R. Ambros

In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees ) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees ) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees ) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates …

Clarification Of The Hybrid Origin Of Carex X Deamii Herm. (Cyperaceae) Based On Macro And Micro Morphological Characters, Paul B. Marcum

Theses, Dissertations and Capstones

The origin of Carex x deamii Herm. has been in question ever since it was named to science in 1938. Collections of the hybrid have always been associated with C. shortiana Dewey and either C. typhina Michx. or C. squarrosa L.. C. typhina and C. squarrosa are closely related taxa (Section Squarrosae) and often are morphologically similar. Because of this similarity, determining the correct parental species to the hybrid has been extremely difficult. It is known that the hybrid is sterile and only reproduces asexually. Pollen was analyzed to ascertain the viability of all four taxa. This study utilizes both …

Developmental Regulation Of A Cyclin-Dependent Kinase Inhibitor Controls Postembryonic Cell Cycle Progression In Caenorhabditis Elegans, Yang Hong, Richard Roy, Victor Ambros

Victor R. Ambros

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in …

Clones Help Develop Ewe Feeding Strategy, Myra Yelland, Rob Kelly, John Davies, Johan Greeff

Journal of the Department of Agriculture, Western Australia, Series 4

Cloned sheep are not a new animal to the researchers of Agriculture WA, but the method that produced Dolly is.

Cloned sheep have been produced at the great Southern Research Institute as early as the mid 1980s. Myra Yelland, Rob Kelly, John Davies and Johan Greef outline how clones are used in experimental studies on wool production

The Cold Shock Domain Protein Lin-28 Controls Developmental Timing In C. Elegans And Is Regulated By The Lin-4 Rna, Eric Moss, Rosalind Lee, Victor Ambros

Victor R. Ambros

Mutations in the heterochronic gene lin-28 of C. elegans cause precocious development where diverse events specific to the second larval stage are skipped. lin-28 encodes a cytoplasmic protein with a cold shock domain and retroviral-type (CCHC) zinc finger motifs, consistent with a role for LIN-28 in posttranscriptional regulation. The 3'UTR of lin-28 contains a conserved element that is complementary to the 22 nt regulatory RNA product of lin-4 and that resembles seven such elements in the 3'UTR of the heterochronic gene lin-14. Both lin-4 activity and the lin-4-complementary element (LCE) are necessary for stage-specific regulation of lin-28. Deleting the LCE …

Heterochronic Genes, Victor Ambros

Victor R. Ambros

This chapter focuses on our current understanding of the C. elegans heterochronic gene pathway as determined by genetic characterization of the regulatory interactions among the genes (Ambros and Horvitz 1987; Ambros 1989; Liu and Ambros 1989) and recent progress on the cloning, sequencing, and molecular analysis of genes involved in this pathway (Ruvkun et al. 1989; Arasu et al. 1991; Papp et al. 1991; Wightman et al. 1991; Lee et al. 1993; Rougvie and Ambros 1995; E. Moss et al., in prep). Primary emphasis is placed on the developmental decisions controlled by the heterochronic genes, the regulatory interactions among genes …

Heterochronic Genes Control Cell Cycle Progress And Developmental Competence Of C. Elegans Vulva Precursor Cells, Susan Euling, Victor Ambros

Victor R. Ambros

Heterochronic genes control the timing of vulval development in the C. elegans hermaphrodite. lin-14 or lin-28 loss-of-function mutations cause the vulval precursor cells (VPCs) to enter S phase and to divide one larval stage earlier than in the wild type. A precocious vulva is formed by essentially normal cell lineage patterns, governed by the same intercellular signals as in the wild type. Mutations that prevent the normal developmental down-regulation of lin-14, activity delay or block VPC division and prevent vulval differentiation. A genetic pathway that includes lin-4, lin-14, and lin-28 controls when VPCs complete G1 and also controls when VPCs …

The Heterochronic Gene Lin-29 Encodes A Zinc Finger Protein That Controls A Terminal Differentiation Event In Caenorhabditis Elegans, Ann Rougvie, Victor Ambros

Victor R. Ambros

A hierarchy of heterochronic genes, lin-4, lin-14, lin-28 and lin-29, temporally restricts terminal differentiation of Caenorhabditis elegans hypodermal seam cells to the final molt. This terminal differentiation event involves cell cycle exit, cell fusion and the differential regulation of genes expressed in the larval versus adult hypodermis. lin-29 is the most downstream gene in the developmental timing pathway and thus it is the most direct known regulator of these diverse processes. We show that lin-29 encodes a protein with five zinc fingers of the (Cys)2-(His)2 class and thus likely controls these processes by regulating transcription in a stage-specific manner. Consistent …

The Caenorhabditis Elegans Heterochronic Gene Pathway Controls Stage-Specific Transcription Of Collagen Genes, Zhongchi Liu, Susan Kirch, Victor Ambros

Victor R. Ambros

In Caenorhabditis elegans, the terminal differentiation of the hypodermal cells occurs at the larval-to-adult molt, and is characterized in part by the formation of a morphologically distinct adult cuticle. The timing of this event is controlled by a pathway of heterochronic genes that includes the relatively direct regulatory gene, lin-29, and upstream genes lin-4, lin-14 and lin-28. Using northern analysis to detect endogenous collagen mRNA levels and collagen/lacZ reporter constructs to monitor collagen transcriptional activity, we show that the stage-specific switch from larval cuticle to adult cuticle correlates with the transcriptional activation of adult-specific collagen genes and repression of larval-specific …

A Thyroid Hormone-Regulated Gene In Xenopus Laevis Encodes A Type Iii Iodothyronine 5-Deiodinase., Donald L. St Germain, Robert Schwartzman, Walburga Croteau, Akira Kanamori, Zhou Wang, Donald D. Brown, Valerie Galton

Dartmouth Scholarship

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in …

Heterochronic Genes And The Temporal Control Of C. Elegans Development, Victor Ambros, Eric Moss

Victor R. Ambros

The heterochronic genes of Caenorhabditis elegans encode part of a regulatory system that controls the temporal component of cell fates in development. The genes have been characterized genetically and molecularly, and their study has so far revealed a genetic hierarchy that specifies sequences of developmental events, a novel RNA-mediated mechanism of gene regulation and a reprogramming phenomenon associated with arrested development.

The C. Elegans Heterochronic Gene Lin-4 Encodes Small Rnas With Antisense Complementarity To Lin-14, Rosalind Lee, Rhonda Feinbaum, Victor Ambros

Victor R. Ambros

lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames …

Efficient Gene Transfer In C.Elegans: Extrachromosomal Maintenance And Integration Of Transforming Sequences, Craig Mello, James Cramer, Dan Stinchcomb, Victor Ambros

Victor R. Ambros

We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can …

Molecular Cloning Of Lin-29, A Heterochronic Gene Required For The Differentiation Of Hypodermal Cells And The Cessation Of Molting In C.Elegans, A. Papp, A. Rougvie, Victor Ambros

Victor R. Ambros

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of …

Heterochronic Genes Control The Stage-Specific Initiation And Expression Of The Dauer Larva Developmental Program In Caenorhabditis Elegans, Zhongchi Liu, Victor R. Ambros

Victor R. Ambros

We report that a stage-specific developmental program, dauer larva formation, is temporally regulated by four heterochronic genes, lin-4, lin-14, lin-28, and lin-29. The effects of mutations in these four genes on dauer larva formation have revealed that they regulate two different processes of dauer larva formation: (1) a decision specifying the larval stage at which dauer larva development initiates, and (2) the specialized differentiation of hypodermal cells during dauer larva morphogenesis. Epistasis analysis has suggested a model in which lin-4 negatively regulates lin-14, and the resulting temporal decrease in lin-14 activity specifies the stage of dauer larva initiation. Our results …

A New Kind Of Informational Suppression In The Nematode Caenorhabditis Elegans, Jonathan Hodgekin, Andrew Papp, Rock Pulak, Victor Ambros, Philip Anderson

Victor R. Ambros

Independent reversions of mutations affecting three different Caenorhabditis elegans genes have each yielded representatives of the same set of extragenic suppressors. Mutations at any one of six loci act as allele-specific recessive suppressors of certain allels of unc-54 (a myosin heavy chain gene), lin-29 (a heterochronic gene), and tra-2 (a sex determination gene). The same mutations also suppress certain alleles of another sex determination gene, tra-1, and of a morphogenetic gene, dpy-5. In addition to their suppression phenotype, the suppressor mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva. We name these genes smg-1 through smg-6 (suppressor …

A Hierarchy Of Regulatory Genes Controls A Larva-To-Adult Developmental Switch In C. Elegans, Victor Ambros

Victor R. Ambros

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific …

Molecular Genetics Of The Caenorhabditis Elegans Heterochronic Gene Lin-14, Gary Ruvkun, Victor Ambros, Alan Coulson, Robert Waterston, John Sulston, H. Horvitz

Victor R. Ambros

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three …

The Lin-14 Locus Of Caenorhabditis Elegans Controls The Time Of Expression Of Specific Postembryonic Developmental Events, Victor R. Ambros, H. Robert Horvitz

Victor R. Ambros

The lin-14 locus of Caenorhabditis elegans plays an important role in specifying the normal timing and sequence of developmental events in the lateral hypodermal cell lineages. The results of gene dosage, complementation, and temperature-shift experiments indicate that the fates expressed by cells at successive stages of these cell lineages are specified by the level of lin-14 activity and that lin-14 acts at multiple times during development to control stage-specific choices of cell fate. Our observations suggest that during normal development a reduction in the level of lin-14 gene function causes the sequential expression of stage-specific cell fates.

The Homeo Domain Of A Murine Protein Binds 5' To Its Own Homeo Box., Abraham Fainsod, Leonard D. Bogarad, Tarmo Ruusala, Martin Lubin

Dartmouth Scholarship

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that …

Heterochronic Mutants Of The Nematode Caenorhabditis Elegans, Victor Ambros, R. Horvitz

Victor R. Ambros

Mutations in the Caenorhabditis elegans genes lin-14, lin-28, and lin-29 cause heterochronic developmental defects: the timing of specific developmental events in several tissues is altered relative to the timing of events in other tissues. These defects result from temporal transformations in the fates of specific cells, that is, certain cells express fates normally expressed by cells generated at other developmental stages. The identification and characterization of genes that can be mutated to cause heterochrony support the proposal that heterochrony is a mechanism for phylogenetic change and suggest cellular and genetic bases for heterochronic variation.

Origin Of Human Trisomy 21 Mosaicism, Diane Dusenbery Waggoner

Dissertations and Theses

Down Syndrome is a human condition caused by an extra copy of a #21 chromosome. At least one to two percent of free (not translocated) trisomy 21 cases are mosaics, i.e., they have two or more distinct cell lines. Usually, one cell line is 47 ,XX or XY ,+21 while the other cell line is normal 46,XX or 46,XY.

The purpose of the study was to establish the etiologies of the separate cell lines by determining whether the zygote was trisomic or normal. Meiotic non-disjunction in the formation of a gamete could lead to a trisomic zygote; loss of a …

Fine Structure Of The Nucleus And Cytoplasmic Feulgen-Positive Areas In The Developing Oocyte Of Argus (Persicargas) Radiatus, Bonnie J. Harding

Biological Sciences Theses & Dissertations

A transmission electron microscope analysis of the changes that occur in the nuclear chromatin and the Feulgen-positive areas of the oocyte cytoplasm as the female develops, feeds and mates is described. The description includes analysis of oocytes from three types of females: unfed, unmated; fed, unmated; and fed, mated. In all types, dense material passes through the extremely porous nuclear membrane where it accumulates in the cytoplasm.

Nuclei of previtellogenic oocytes appear the same in all females studied. Fibrillar bodies and up to four large nucleoli are seen. No typical chromatin is seen, but structures resembling nucleosomes appear throughout the …

More Lambs By Mating Weaners, R J. Suiter

Journal of the Department of Agriculture, Western Australia, Series 4

Farmers can increase sheep numbers by mating ewe weaners. Trials have shoen that this does not affect the later performance of these ewes.

Later Mating Improves Lambing Results, R J. Suiter

Journal of the Department of Agriculture, Western Australia, Series 4

Ovulation studies in Western Australia have indicated that increased lambing percentages in crossbred and Merino ewes could be expected from mating in February-April rather than December- January.

Such increases have been demonstrated in time of lambing trials with crossbred ewes, but had not been seen in Merino flocks until recently.

This report summarises the results of time of lambing trials with Merinos carried out at the Merredin and Wongan Hills Research Stations between 1963 and 1969.