Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

scholarly journals Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948 ◽  
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

Synthesis of a ribosome-bound translatable poly(A)− mRNA during spore germination in Dictyostelium discoideum

1989 ◽  
Vol 35 (5) ◽  
pp. 573-577
Author(s):  
S. Ramagopal
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Gel Electrophoresis ◽  
Rna Synthesis ◽  
In Vitro Translation ◽  
Cellular Slime Mold ◽  
Free System ◽  
Cellular Slime

A distinct poly(A)− RNA sedimenting around 10–12S was identified during spore germination in Dictyostelium discoideum. Activated spores were labeled with [3H]uracil and the poly(A)− RNA was purified from ribosomal particles for analysis. In the spore swelling stage, 40 to 50% of the newly synthesized poly(A)− RNA was 10–12S RNA. This fraction diminished to one-half or one-fourth depending on the labeling period at the stage of amoeba emergence. The 10–12S RNA was associated with both monosomes and polysomes in vivo. Translation in a wheat germ cell-free system and gel electrophoresis demonstrated that the 10–12S RNA coded for a number of polypeptides, some of which were also represented among the in vitro products of poly(A)+ RNA. However, there were seven unique polypeptides (37.5, 28.2, 27.5, 23, 17.7, 17, and 14.2 kilodaltons) encoded exclusively by 10–12S RNA.Key words: cellular slime mold, RNA synthesis, development, poly(A)− mRNA, in vitro translation.

scholarly journals Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2142-2150 ◽  
Author(s):  
R A Levine ◽  
G J LaRosa ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Cdna Library ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Cdna Clones ◽  
Dibutyryl Cyclic Amp

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA

1990 ◽  
Vol 10 (5) ◽  
pp. 2315-2326
Author(s):  
J H Teruya ◽  
S Y Kutsunai ◽  
D H Spear ◽  
P A Edwards ◽  
C F Clarke
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Farnesyl Pyrophosphate ◽  
S1 Nuclease ◽  
Testis Cdna ◽  
Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product

1985 ◽  
Vol 5 (4) ◽  
pp. 816-822
Author(s):  
H J Himmelfarb ◽  
E Maicas ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Blot Hybridization ◽  
Single Copy ◽  
Suppressor Gene ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Nonsense Suppression ◽  
Synthesis Rate

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

scholarly journals Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA.

1990 ◽  
Vol 10 (5) ◽  
pp. 2315-2326 ◽  
Author(s):  
J H Teruya ◽  
S Y Kutsunai ◽  
D H Spear ◽  
P A Edwards ◽  
C F Clarke
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Farnesyl Pyrophosphate ◽  
S1 Nuclease ◽  
Testis Cdna ◽  
Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

The hyaluronate lyase of Staphylococcus aureus – a virulence factor?

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 2005-2013 ◽  
Author(s):  
George Makris ◽  
John D. Wright ◽  
Eileen Ingham ◽  
Keith T. Holland
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Colorimetric Assay ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Temperature Sensitive ◽  
Cell Recovery ◽  
Hyaluronate Lyase

The hyaluronate lyase (HL) gene of Staphylococcus aureus 8325-4 (hysA) was inactivated in vitro with the insertion of the erythromycin determinant, ermC, from plasmid pE194. The hysA : : ermC mutation was introduced into S. aureus via a temperature-sensitive shuttle vector, where it underwent homologous recombination with the wild-type (w.t.) allele. The insertion of ermC in the chromosomal hysA locus was confirmed by Southern blot hybridization and the loss of HL activity was demonstrated macroscopically by a plate assay. The importance of HL for pathogenicity was assessed by comparing the virulence of the HL− mutant strain to that of the w.t. in an established mouse abscess model of S. aureus infection. A significantly higher cell recovery was obtained from lesions infected with the w.t. strain compared to the lesions infected with the HL− strain (P =0·01). Although the lesion areas from both groups were not significantly different (P=0·9) they were of different morphology. A colorimetric assay was used to measure HL activity from culture supernatants of the S. aureus 8325-4 strains w.t., WA250 (agr) and PC1839 (sar) grown in a chemically defined medium. HL activity reached a maximum in the w.t. strain during mid-exponential phase (t=5 h) and while it showed a 16-fold decrease in the agr mutant it increased 35-fold in the sar mutant background. These results strongly suggest that HL is a virulence factor which is important in the early stages of subcutaneous infections.

scholarly journals Influence of Mouse-Strain-Specific Factors on Position-Dependent Transgene DNA Methylation Patterns

1996 ◽  
Vol 45 (1-2) ◽  
pp. 243-244
Author(s):  
P.A. Koetsier ◽  
W. Doerfler
Keyword(s):  
Dna Methylation ◽  
Genetic Background ◽  
Promoter Activity ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Adenovirus Type ◽  
Methylation Pattern ◽  
Parent Of Origin ◽  
Methylation Patterns

In previous work from this laboratory, an inverse dependence was established for the adenovirus type 2 E2A late promoter between sequence-specific DNA methylation and promoter activity [1-5; for reviews see ref. 6, 7]. The effect of DNA methylation on promoter activity was also assessed in the transgenic mice, which were obtained from microinjections of unmethylated or in vitro HpaII-premethylated pAd2E2AL-CAT DNA [1] into F2 zygotes from B6D2F, (C57BL/6 × DBA/2) hybrid mice. In CAT assays carried out on organ extracts from the pAd2E2AL-CAT mice, the inverse relationship was confirmed [2].We studied the stability of the pAd2E2AL-CAT DNA methylation patterns in up to eight mouse generations and assessed the influence of the strain-specific genetic background. Three pAd2E2AL-CAT mouse lines were crossed with inbred DBA/2, C57BL/6 or B6D2F, mice. Parent-of-origin effects were controlled by exclusive hemizygous transgene transmission either via females or males. The founder animal of line 7-1 carried two groups of transgenes (A and B) on separate chromosomes. The transgene methylation patterns of the 7-1B transgenes and those of the lines 5-8 and 8-1 were stably transmitted.Southern blot hybridization experiments [8, 9] revealed that the 7-1A transgene methylation pattern was a cellular mosaic. In mixed-genetic-background offspring from 7-1A animals, 10% carried transgenes with HpaII-DNA methylation levels that were reduced from 40 to 10-15%. This finding suggested that in this background the factors that supported high methylation levels were dominant. When inbred DBA/2 mice were the mates, 40% of the siblings carried demethylated transgenes, whereas this ratio amounted to only 10% in C57BL/6 offspring (comparable to B6D2F1 crossings). Transgene methylation patterns were not detectably influenced by the parent-of-origin.

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