scholarly journals Novel insertion mutation in Caenorhabditis elegans.

1985 ◽  
Vol 5 (1) ◽  
pp. 1-6 ◽  
Author(s):  
D J Eide ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Base Pair ◽  
Alkylating Agent ◽  
Body Wall ◽  
Insertion Site ◽  
Direct Repeat ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Homologous Sequences ◽  
Pair Sequence

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.

Novel insertion mutation in Caenorhabditis elegans

1985 ◽  
Vol 5 (1) ◽  
pp. 1-6
Author(s):  
D J Eide ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Base Pair ◽  
Alkylating Agent ◽  
Body Wall ◽  
Insertion Site ◽  
Direct Repeat ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Homologous Sequences ◽  
Pair Sequence

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells

1990 ◽  
Vol 10 (1) ◽  
pp. 113-119
Author(s):  
F L Lin ◽  
K Sperle ◽  
N Sternberg
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Insertion Site ◽  
Strand Break ◽  
Insertion Mutation ◽  
Dna Breaks ◽  
Base Pairs ◽  
Restriction Sites ◽  
Single Strand Annealing ◽  
Strand Annealing

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.

Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes

1989 ◽  
Vol 9 (3) ◽  
pp. 1365-1367
Author(s):  
D S Ray
Keyword(s):  
Base Pair ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Kinetoplast Dna ◽  
Base Pairs ◽  
Conserved Sequence ◽  
Diverse Species ◽  
Pair Sequence ◽  
Sequence Region ◽  
Minicircle Sequence

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.

scholarly journals Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes.

1989 ◽  
Vol 9 (3) ◽  
pp. 1365-1367 ◽  
Author(s):  
D S Ray
Keyword(s):  
Base Pair ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Kinetoplast Dna ◽  
Base Pairs ◽  
Conserved Sequence ◽  
Diverse Species ◽  
Pair Sequence ◽  
Sequence Region ◽  
Minicircle Sequence

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.

scholarly journals Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

1990 ◽  
Vol 10 (1) ◽  
pp. 113-119 ◽  
Author(s):  
F L Lin ◽  
K Sperle ◽  
N Sternberg
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Insertion Site ◽  
Strand Break ◽  
Insertion Mutation ◽  
Dna Breaks ◽  
Base Pairs ◽  
Restriction Sites ◽  
Single Strand Annealing ◽  
Strand Annealing

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Biochemistry ◽  
1990 ◽  
Vol 29 (18) ◽  
pp. 4446-4456 ◽  
Author(s):  
Elizabeth T. Zuo ◽  
Farial A. Tanious ◽  
W. David Wilson ◽  
Gerald Zon ◽  
Guo Shen Tan ◽  
...  
Keyword(s):  
Base Pair ◽  
Base Pairs ◽  
Thermally Induced ◽  
Pair Sequence

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

scholarly journals A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 483-498
Author(s):  
J Ahnn ◽  
A Fire
Keyword(s):  
Caenorhabditis Elegans ◽  
Myosin Heavy Chain ◽  
Muscle Cell ◽  
Heavy Chain ◽  
Body Wall ◽  
Muscle Development ◽  
Contractile Function ◽  
The Body ◽  
Body Wall Muscle ◽  
Genetic Loci

Abstract We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.

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