scholarly journals Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 501-516 ◽  
Author(s):  
G Ruvkun ◽  
V Ambros ◽  
A Coulson ◽  
R Waterston ◽  
J Sulston ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
General Strategy ◽  
Cis Acting ◽  
Temporal Regulation ◽  
C Elegans ◽  
Dna Alterations ◽  
Heterochronic Gene ◽  
Systematic Identification

Abstract 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 closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.

scholarly journals Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice

1993 ◽  
Vol 90 (23) ◽  
pp. 11381-11385 ◽  
Author(s):  
K M Gaensler ◽  
M Kitamura ◽  
Y W Kan
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Physiological Role ◽  
Artificial Chromosome ◽  
Chromatin Domain ◽  
Globin Genes ◽  
Beta Globin ◽  
Cis Acting ◽  
Artificial Chromosomes

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

scholarly journals Multiple, Distant Gata2 Enhancers Specify Temporally and Tissue-Specific Patterning in the Developing Urogenital System

2004 ◽  
Vol 24 (23) ◽  
pp. 10263-10276 ◽  
Author(s):  
Melin Khandekar ◽  
Norio Suzuki ◽  
Jon Lewton ◽  
Masayuki Yamamoto ◽  
James Douglas Engel
Keyword(s):  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
General Strategy ◽  
Urogenital System ◽  
Loss Of Function ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Null Mutant Mice

ABSTRACT Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3′ to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

Construction and analysis of artificial chromosomes with de novo holocentromeres in Caenorhabditis elegans

2020 ◽  
Vol 64 (2) ◽  
pp. 233-249
Author(s):  
Zhongyang Lin ◽  
Karen Wing Yee Yuen
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Sequence ◽  
De Novo ◽  
Complex Mixture ◽  
Chromosome Length ◽  
Cis Acting ◽  
Artificial Chromosomes ◽  
C Elegans ◽  
Successive Generations

Abstract Artificial chromosomes (ACs), generated in yeast (YACs) and human cells (HACs), have facilitated our understanding of the trans-acting proteins, cis-acting elements, such as the centromere, and epigenetic environments that are necessary to maintain chromosome stability. The centromere is the unique chromosomal region that assembles the kinetochore and connects to microtubules to orchestrate chromosome movement during cell division. While monocentromeres are the most commonly characterized centromere organization found in studied organisms, diffused holocentromeres along the chromosome length are observed in some plants, insects and nematodes. Based on the well-established DNA microinjection method in holocentric Caenorhabditis elegans, concatemerization of foreign DNA can efficiently generate megabase-sized extrachromosomal arrays (Exs), or worm ACs (WACs), for analyzing the mechanisms of WAC formation, de novo centromere formation, and segregation through mitosis and meiosis. This review summarizes the structural, size and stability characteristics of WACs. Incorporating LacO repeats in WACs and expressing LacI::GFP allows real-time tracking of newly formed WACs in vivo, whereas expressing LacI::GFP-chromatin modifier fusions can specifically adjust the chromatin environment of WACs. The WACs mature from passive transmission to autonomous segregation by establishing a holocentromere efficiently in a few cell cycles. Importantly, WAC formation does not require any C. elegans genomic DNA sequence. Thus, DNA substrates injected can be changed to evaluate the effects of DNA sequence and structure in WAC segregation. By injecting a complex mixture of DNA, a less repetitive WAC can be generated and propagated in successive generations for DNA sequencing and analysis of the established holocentromere on the WAC.

Muscle and nerve-specific regulation of a novel NK-2 class homeodomain factor in Caenorhabditis elegans

Development ◽  
1998 ◽  
Vol 125 (3) ◽  
pp. 421-429 ◽  
Author(s):  
B.D. Harfe ◽  
A. Fire
Keyword(s):  
Caenorhabditis Elegans ◽  
Motor Neurons ◽  
Homeobox Gene ◽  
Cell Types ◽  
Egg Laying ◽  
Cis Acting ◽  
C Elegans ◽  
E Box ◽  
Vulval Muscle ◽  
Specific Regulation

We have identified a new Caenorhabditis elegans NK-2 class homeobox gene, designated ceh-24. Distinct cis-acting elements generate a complex neuronal and mesodermal expression pattern. A promoter-proximal enhancer mediates expression in a single pharyngeal muscle, the donut-shaped m8 cell at the posterior end of the pharynx. A second mesodermal enhancer is active in a set of eight nonstriated vulval muscles used in egg laying. Activation in the egg laying muscles requires an ‘NdE-box’ consensus motif (CATATG) which is related to, but distinct from, the standard E-box motif bound by the MyoD family of transcriptional activators. Ectodermal expression of ceh-24 is limited to a subset of sublateral motor neurons in the head of the animal; this activity requires a cis-acting activator element that is distinct from the control elements for pharyngeal and vulval muscle expression. Activation of ceh-24 in each of the three cell types coincides with the onset of differentiation. Using a set of transposon-induced null mutations, we show that ceh-24 is not essential for the formation of any of these cells. Although ceh-24 mutants have no evident defects under laboratory conditions, the pattern of ceh-24 activity is apparently important for Rhabditid nematodes: the related species C. briggsae contains a close homologue of C. elegans ceh-24 including a highly conserved and functionally equivalent set of cis-acting control signals.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

scholarly journals Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

2008 ◽  
Vol 19 (5) ◽  
pp. 2154-2168 ◽  
Author(s):  
Corey L. Williams ◽  
Marlene E. Winkelbauer ◽  
Jenny C. Schafer ◽  
Edward J. Michaud ◽  
Bradley K. Yoder
Keyword(s):  
Caenorhabditis Elegans ◽  
Joubert Syndrome ◽  
Cystic Kidney ◽  
Basal Bodies ◽  
The Novel ◽  
C Elegans ◽  
Human Genes ◽  
Cilia Formation ◽  
Strong Candidate ◽  
Candidate Loci

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

Maternal-effect lethal mutations on linkage group II of Caenorhabditis elegans.

Genetics ◽  
1988 ◽  
Vol 120 (4) ◽  
pp. 977-986
Author(s):  
K J Kemphues ◽  
M Kusch ◽  
N Wolf
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Maternal Effect ◽  
Essential Genes ◽  
The Other ◽  
C Elegans ◽  
Lethal Mutations ◽  
Embryonic Lethal ◽  
Embryonic Lethal Mutations ◽  
F1 Progeny

Abstract We have analyzed a set of linkage group (LG) II maternal-effect lethal mutations in Caenorhabditis elegans isolated by a new screening procedure. Screens of 12,455 F1 progeny from mutagenized adults resulted in the recovery of 54 maternal-effect lethal mutations identifying 29 genes. Of the 54 mutations, 39 are strict maternal-effect mutations defining 17 genes. These 17 genes fall into two classes distinguished by frequency of mutation to strict maternal-effect lethality. The smaller class, comprised of four genes, mutated to strict maternal-effect lethality at a frequency close to 5 X 10(-4), a rate typical of essential genes in C. elegans. Two of these genes are expressed during oogenesis and required exclusively for embryogenesis (pure maternal genes), one appears to be required specifically for meiosis, and the fourth has a more complex pattern of expression. The other 13 genes were represented by only one or two strict maternal alleles each. Two of these are identical genes previously identified by nonmaternal embryonic lethal mutations. We interpret our results to mean that although many C. elegans genes can mutate to strict maternal-effect lethality, most genes mutate to that phenotype rarely. Pure maternal genes, however, are among a smaller class of genes that mutate to maternal-effect lethality at typical rates. If our interpretation is correct, we are near saturation for pure maternal genes in the region of LG II balanced by mnC1. We conclude that the number of pure maternal genes in C. elegans is small, being probably not much higher than 12.

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