scholarly journals Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

1986 ◽  
Vol 6 (1) ◽  
pp. 142-149 ◽  
Author(s):  
M B Kurtz ◽  
M W Cortelyou ◽  
D R Kirsch
Keyword(s):  
Candida Albicans ◽  
Dna Sequences ◽  
Southern Hybridization ◽  
Sexual Cycle ◽  
Dna Transformation ◽  
Common Region ◽  
Integrative Transformation ◽  
Dimorphic Yeast ◽  
Ade2 Gene ◽  
Yeast Plasmids

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene

1986 ◽  
Vol 6 (1) ◽  
pp. 142-149
Author(s):  
M B Kurtz ◽  
M W Cortelyou ◽  
D R Kirsch
Keyword(s):  
Candida Albicans ◽  
Dna Sequences ◽  
Southern Hybridization ◽  
Sexual Cycle ◽  
Dna Transformation ◽  
Common Region ◽  
Integrative Transformation ◽  
Dimorphic Yeast ◽  
Ade2 Gene ◽  
Yeast Plasmids

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces

Genome ◽  
1988 ◽  
Vol 30 (5) ◽  
pp. 690-696 ◽  
Author(s):  
Wendy H. Horsfall ◽  
Ronald E. Pearlman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Assay System ◽  
Mitotic Stability ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genomic Libraries ◽  
Ade2 Gene

Genomic libraries containing micronuclear DNA sequences from Tetrahymena thermophila have been constructed in a vector containing ARS1, SUP11, and ura3 sequences from the yeast Saccharomyces cerevisiae. When transformed into a strain of S. cerevisiae carrying a suppressible ochre mutation in the ade2 gene, viable transformants are obtained only if the transforming plasmid is maintained at a copy number of one or two per cell. Mitotic segregation of the plasmid is easily assessed in a colour assay of transformants. Using this assay system, we showed that micronuclear DNA from Tetrahymena does not contain sequences that confer mitotic stability on yeast ARS-containing plasmids; i.e., sequences that function analogously to yeast centromere sequences. One transformant was analyzed that carries Tetrahymena sequences that maintain the copy number of the ARS plasmid at one or two per cell. However, these sequences do not confer mitotic stability on the transformants and they confer a phenotype in this assay similar to that of the REP3 gene of the yeast 2 μm plasmid.Key words: mitotic stability, centromere, Tetrahymena, Saccharomyces.

scholarly journals Retention of relict satellite DNA sequences in Anemone (Ranunculaceae)

2013 ◽  
Vol 72 (1) ◽  
pp. 1-133 ◽  
Author(s):  
Višnja Besendorfer ◽  
Jelena Mlinarec
Keyword(s):  
Dna Sequences ◽  
Southern Hybridization ◽  
Repeat Unit ◽  
Similar Species ◽  
Genomic Component ◽  
Library Hypothesis ◽  
Species Specific ◽  
Eukaryotic Organisms ◽  
Fish Signal

Abstract Satellite DNAis a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNAis an important element in genome organization and evolution in plants. Here we study the presence, physical distribution and abundance of the satellite DNAfamily AhTR1 in Anemone. Twenty-two Anemone accessions were analyzed by PCR to assess the presence of AhTR1, while fluorescence in situ hybridization and Southern hybridization were used to determine the abundance and genomic distribution of AhTR1. The AhTR1 repeat unit was PCR-amplified only in eight phylogenetically related European Anemone taxa of the Anemone section. FISH signal with AhTR1 probe was visible only in A. hortensis and A. pavonina, showing localization of AhTR1 in the regions of interstitial heterochromatin in both species. The absence of a FISH signal in the six other taxa as well as weak signal after Southern hybridization suggest that in these species AhTR1 family appears as relict sequences. Thus, the data presented here support the »library hypothesis« for AhTR1 satellite evolution in Anemone. Similar species-specific satellite DNAprofiles in A. hortensis and A. pavonina support the treatment of A. hortensis and A. pavonina as one species, i.e. A. hortensis s.l.

Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants

1987 ◽  
Vol 7 (1) ◽  
pp. 199-208
Author(s):  
R Kelly ◽  
S M Miller ◽  
M B Kurtz ◽  
D R Kirsch
Keyword(s):  
Candida Albicans ◽  
Gene Disruption ◽  
Orotic Acid ◽  
Deletion Mutant ◽  
Southern Hybridization ◽  
Restriction Site ◽  
Insertion Mutant ◽  
Ura3 Gene ◽  
Insertional Inactivation ◽  
One Step

A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.

scholarly journals High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 1093-1103 ◽  
Author(s):  
J T Irelan ◽  
A T Hagemann ◽  
E U Selker
Keyword(s):  
Point Mutation ◽  
Dna Sequences ◽  
High Frequency ◽  
Recombination Frequency ◽  
Single Copy ◽  
Sexual Cycle ◽  
Base Pairs ◽  
Copy Gene ◽  
The Relationship ◽  
Repeat Induced Point Mutation

Abstract Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.

The dependency of nuclear division on volume in the dimorphic yeast Candida albicans

1981 ◽  
Vol 133 (1) ◽  
pp. 55-62 ◽  
Author(s):  
David R. Soll ◽  
Glenn Bedell ◽  
Julie Thiel ◽  
Marvin Brummel
Keyword(s):  
Candida Albicans ◽  
Nuclear Division ◽  
Dimorphic Yeast

scholarly journals Variation of the non-factor VIII sequences detected by a probe from intron 22 of the factor VIII gene

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 139-143 ◽  
Author(s):  
DP Lillicrap ◽  
SA Taylor ◽  
PC Schuringa ◽  
VS Blanchette ◽  
JK Lovsted ◽  
...  
Keyword(s):  
Factor Viii ◽  
Dna Sequences ◽  
Southern Hybridization ◽  
Hemophilia A ◽  
Direct Analysis ◽  
Variant Form ◽  
Partial Deletion ◽  
Factor Viii Gene ◽  
New Variant ◽  
Xbai Polymorphism

Abstract A severe hemophilia A family has been studied with the factor VIII (F.VIII) intragenic XbaI polymorphism. During this investigation, a new variant hybridization pattern was observed with important implications concerning the non-F.VIII DNA sequences detected by the probe from intron 22, p482.6. Both Southern hybridization studies and direct analysis of amplified DNA demonstrated a variant form of the non-F.VIII sequences. This variant DNA sequence has not been responsible for any detectable phenotypic abnormalities, and likely represents a polymorphic change. In conclusion, this study has shown that the non- F.VIII sequences detected with the probe p482.6 are situated on the X chromosome, they seem to be present in two copies, and either or both copies infrequently possess a polymorphic XbaI site or a partial deletion.

PstI repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo

Genome ◽  
2002 ◽  
Vol 45 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Faruk G Sheikh ◽  
Sudit S Mukhopadhyay ◽  
Prabhakar Gupta
Keyword(s):  
Nucleotide Sequence ◽  
Dna Sequences ◽  
Sequence Homology ◽  
Copy Number ◽  
Southern Hybridization ◽  
Repetitive Dna Sequences ◽  
Type I ◽  
Type Ii ◽  
Repeat Type ◽  
Highly Repetitive Dna

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 × 105 – 4 × 105, and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.Key words: repeat, SINE, Bovidae, genome.

scholarly journals Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

1986 ◽  
Vol 6 (5) ◽  
pp. 1520-1528 ◽  
Author(s):  
D Y Chang ◽  
B Wisely ◽  
S M Huang ◽  
R A Voelker
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Insertion Site ◽  
Hybrid Dysgenesis ◽  
P Element ◽  
Dna Transformation ◽  
Wild Type ◽  
Induced Mutations ◽  
X Ray ◽  
Element Insertion

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

1981 ◽  
Vol 27 (6) ◽  
pp. 580-585 ◽  
Author(s):  
Louise A. Brown ◽  
W. LaJean Chaffin
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Germ Tube ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tube Formation ◽  
Yeast Cells ◽  
Cytoplasmic Proteins ◽  
Specific Proteins ◽  
Major Shift ◽  
Dimorphic Yeast

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 °C, form budding yeast and at 37 °C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.

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