scholarly journals Analysis of the Caenorhabditis elegans axonal guidance and outgrowth gene unc-33.

Genetics ◽  
1992 ◽  
Vol 132 (3) ◽  
pp. 675-689 ◽  
Author(s):  
W Li ◽  
R K Herman ◽  
J E Shaw
Keyword(s):  
Caenorhabditis Elegans ◽  
Gamma Ray ◽  
Protein A ◽  
Genomic Region ◽  
Axonal Guidance ◽  
Significant Similarity ◽  
Spliced Leader ◽  
Neuronal Processes ◽  
Type Size ◽  
Intermediate Size

Abstract Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.

scholarly journals Molecular and genetic analysis of unc-7, a Caenorhabditis elegans gene required for coordinated locomotion.

Genetics ◽  
1993 ◽  
Vol 133 (3) ◽  
pp. 527-541 ◽  
Author(s):  
T A Starich ◽  
R K Herman ◽  
J E Shaw
Keyword(s):  
Caenorhabditis Elegans ◽  
Motor Neurons ◽  
Gamma Ray ◽  
Developmental Stages ◽  
Northern Analysis ◽  
Wild Type ◽  
Spliced Leader ◽  
Translational Start Site ◽  
Translational Start ◽  
Tc1 Element

Abstract Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.

Regulatory myosin light-chain genes of Caenorhabditis elegans

1988 ◽  
Vol 8 (12) ◽  
pp. 5339-5349
Author(s):  
C Cummins ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Light Chain ◽  
Calcium Binding ◽  
Myosin Light Chain ◽  
Genomic Region ◽  
Leader Sequence ◽  
Light Chains ◽  
Spliced Leader ◽  
C Elegans ◽  
Regulatory Myosin Light Chain

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.

scholarly journals Regulatory myosin light-chain genes of Caenorhabditis elegans.

1988 ◽  
Vol 8 (12) ◽  
pp. 5339-5349 ◽  
Author(s):  
C Cummins ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Light Chain ◽  
Calcium Binding ◽  
Myosin Light Chain ◽  
Genomic Region ◽  
Leader Sequence ◽  
Light Chains ◽  
Spliced Leader ◽  
C Elegans ◽  
Regulatory Myosin Light Chain

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering

Genome ◽  
2017 ◽  
Vol 60 (6) ◽  
pp. 537-545 ◽  
Author(s):  
Kumiko Ui-Tei ◽  
Shohei Maruyama ◽  
Yuko Nakano
Keyword(s):  
Rna Polymerase Iii ◽  
Protein A ◽  
Genomic Region ◽  
Guide Rna ◽  
Genomic Engineering ◽  
System A ◽  
Double Stranded Dna ◽  
Transcriptional Termination ◽  
Sequence Complementarity ◽  
Pol Iii

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

scholarly journals The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 987-997 ◽  
Author(s):  
S G Clark ◽  
X Lu ◽  
H R Horvitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
Genomic Region ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Intercellular Signaling ◽  
Tyrosine Kinase Signaling ◽  
Genomic Regions ◽  
Mrna Precursor

Abstract The Caenorhabditis elegans locus lin-15 negatively regulates an intercellular signaling process that induces formation of the hermaphrodite vulva. The lin-15 locus controls two separate genetic activities. Mutants that lack both activities have multiple, ectopic pseudo-vulvae resulting from the overproduction of vulval cells, whereas mutants defective in only one lin-15 activity appear wild-type. lin-15 acts non-cell-autonomously to prevent the activation of a receptor tyrosine kinase/ras signaling pathway. We report here the molecular characterization of the lin-15 locus. The two lin-15 activities are encoded by contiguous genomic regions and by two distinct, non-overlapping transcripts that may be processed from a single mRNA precursor by trans-splicing. Based on the DNA sequence, the 719- and 1,440-amino acid lin-15 proteins are not similar to each other or to known proteins. lin-15 multivulva mutants, which are defective in both lin-15 activities, contain deletions and insertions that affect the lin-15 genomic region.

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum

1989 ◽  
Vol 9 (8) ◽  
pp. 3543-3547
Author(s):  
T W Nilsen ◽  
J Shambaugh ◽  
J Denker ◽  
G Chubb ◽  
C Faser ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Secondary Structure ◽  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Ascaris Lumbricoides ◽  
Parasitic Nematode ◽  
Secondary Structures ◽  
Spliced Leader ◽  
C Elegans

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

Many transcribed regions of the Onchocerca volvulus genome contain the spliced leader sequence of Caenorhabditis elegans

1990 ◽  
Vol 10 (6) ◽  
pp. 2765-2773
Author(s):  
W L Zeng ◽  
C M Alarcon ◽  
J E Donelson
Keyword(s):  
Caenorhabditis Elegans ◽  
Repeat Unit ◽  
5S Rrna ◽  
Onchocerca Volvulus ◽  
Rrna Genes ◽  
Free Living ◽  
Spliced Leader ◽  
C Elegans ◽  
Perfect Sequence ◽  
5S Rrna Genes

Genomic DNAs of the related parasitic nematodes Onchocerca volvulus and Dirofilariae immitis, and a cDNA library of O. volvulus, were examined for the presence of the 22-nucleotide spliced leader (SL) found at the 5' ends of 10 to 15% of the mRNAs in the free-living nematode Caenorhabditis elegans. As in C. elegans, genes for the SL RNA are linked to the repetitive 5S rRNA genes of O. volvulus and D. immitis, but unlike C. elegans, they are in the same orientation as the 5S rRNA genes within the repeat unit. In O. volvulus the SL sequence is also encoded at more than 30 additional genomic locations and occurs at interior sites within many transcripts. Sequence determinations of four different cDNAs of O. volvulus, each containing an internal copy of the SL within a conserved 25mer, and one corresponding genomic DNA clone indicate that this sequence is not trans spliced onto these RNAs, but is encoded within the genes. The RNAs of two of these cDNAs appear to be developmentally regulated, since they occur in adult O. volvulus but were not detected in the infective L3 stage larvae. In contrast, actin mRNAs are present at all developmental stages, and at least one actin mRNA species contains a trans-spliced 5' SL. The internal locations of the SL in various transcripts and its perfect sequence conservation among parasitic and free-living nematodes argues that it serves specific, and perhaps multiple, functions for these organisms.

scholarly journals An ankyrin-related gene (unc-44) is necessary for proper axonal guidance in Caenorhabditis elegans.

1995 ◽  
Vol 129 (4) ◽  
pp. 1081-1092 ◽  
Author(s):  
A J Otsuka ◽  
R Franco ◽  
B Yang ◽  
K H Shim ◽  
L Z Tang ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nucleotide Sequence ◽  
Axon Guidance ◽  
Axonal Guidance ◽  
Related Gene ◽  
Sequence Analyses ◽  
Molecular Analyses ◽  
Molecular Studies ◽  
Alternatively Spliced ◽  
Related Proteins

Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AO13 ankyrin (< or = 4700 aa, < or = 517 kD). In addition to the major set of approximately 6 kb alternatively spliced transcripts, minor transcripts were observed at approximately 3, 5, 7, and 14 kb. Evidence is provided that mutations in the approximately 14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.

Analysis of lethal mutations induced in a mutator strain that activates transposable elements in Caenorhabditis elegans

Genome ◽  
1990 ◽  
Vol 33 (1) ◽  
pp. 109-114 ◽  
Author(s):  
Denise V. Clark ◽  
Robert C. Johnsen ◽  
Kim S. McKim ◽  
David L. Baillie
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Gamma Ray ◽  
Hot Spot ◽  
Single Gene ◽  
Gene Mutations ◽  
Group Iv ◽  
Group V ◽  
Mutator Strain ◽  
Lethal Mutations

A screen was conducted for lethal mutations in the nematode Caenorhabditis elegans in a strain containing the mutator mut-4(st700)I to examine the nature of mutator-induced lethal mutations within two large chromosomal regions comprising a total of 49 map units (linkage group IV (right) and linkage group V (left)). The genetic analysis of 28 lethal mutations has revealed that the mutator locus mut-4(st700)I causes both putative single-gene mutations and deficiencies. We have identified lethal mutations in three different genes, in addition to seven deficiencies. There is a mutational hot spot on linkage group V (left) around the lin-40 locus. Six mutations appear to be alleles of lin-40. In addition, 5 of 7 deficiencies have breakpoints at or very near lin-40. All seven deficiencies delete the left-most known gene on linkage group V (left) and thus appear to delete the tip of the chromosome. This is in contrast to gamma ray and formaldehyde induced deficiencies, which infrequently delete the closest known gene to the tip of a chromosome.Key words: Caenorhabditis elegans, mutator, deficiencies, lethal mutations.

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