Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition

Mangiarotti, G; Chung, Sungwon; Zuker, Charles S.; Lodish, Harvey F.

doi:10.1093/nar/9.4.947

Cited by 85 publications

(46 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In Further complications may arise from repeat sequences in different mRNAs. These have been shown to exist in slime molds (18,41) and possibly affect the selection procedures (22). Our selection procedures included higher criteri- Quantitative measurements of the steadystate concentrations showed that individual pluteus-specific mRNAs accumulated to different extents.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Fregien¹,

Dolecki²,

Mandel³

et al. 1983

Mol. Cell. Biol.

View full text Add to dashboard Cite

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3-and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.The sea urchin embryo appears to be one of the most accessible developmental systems for studying the molecular events of cell type determination during early embryogenesis. After fertilization, eggs divide into a number of cells whose developmental fate is fixed somewhat before they differentiate into the various cell types. Most of the cells in the sea urchin embryo contribute to the three layers of the relatively simple pluteus larva: the ectodermal covering, the skeletal-forming mesenchyme, and the gut. These differentiated tissues function during the 1 to several months of larval life. The actual sea urchin emerges at metamorphosis after it has grown from a few cells set aside during embryogenesis for its development.Despite the simplicity of the system and the numerous molecular studies on sea urchin embryos, these larval tissues have not received much attention as specialized cells synthesizing cell type-specific proteins. The major reason has been that there is no evidence of very abundant With the advent of molecular cloning, it has become possible to define, isolate, and study genes coding for proteins produced only in limited abundance, including putative specialized proteins of the three cell layers of the pluteus. Clones of mRNA and genomic sequences which code for sev...

show abstract

Section: Discussionmentioning

confidence: 99%

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Fregien¹,

Dolecki²,

Mandel³

et al. 1983

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…Labeling of DNA (nick-translation), gel fractionation of RNA and DNA, and blotting onto nitrocellulose filters were performed as described (7,8). Prehybridization, hybridization, and washing of filters were conducted exactly as described (5).…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of Genomic Clones. The Dictyostelium EcoRI genomic library in A gt-XWEs has been described (8). The Charon 28 library was constructed by ligating partially digested Sau3A genomic framents to the BamHI site of the vector (9) and will be described in detail elsewhere (unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Dictyostelium transposable element DIRS-1 has 350-base-pair inverted terminal repeats that contain a heat shock promoter.

Zuker¹,

Cappello²,

Lodish³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

DIRS-1 is a 4.7-kilobase-pair repetitive and apparently transposable Dictyostelium genetic element that is transcribed during differentiation or after heat shock. The terminal regions of DIRS-1 are inverted repeats of 330 base pairs. The repeats are highly conserved both within a given element as well as between different members of the family (<10% divergence). At the distal end of all left repeats is a 32-nucleotide sequence composed almost entirely of A and T residues. In addition to this 32-base A+T sequence, the distal region of all right repeats is extended by a 28-base-pair A+T-rich sequence that is identical in all copies. The sequences flanking each DIRS-1 sequence are completely dissimilar, and there appears to be no duplication of the genomic DNA sequence at the presumed point of DIRS-1 insertion. The terminal repeats can also be found interspersed in the genome independently of the complete element. In addition, the terminal repeats carry a 15-nucleotide sequence that greatly resembles the Drosophila consensus heat shock promoter and may be involved in the transcriptional induction of the DIRS-1 sequences. A common feature of most eukaryotic and prokaryotic transposable elements is the presence of terminally repeated sequences-either direct (copia, Ty-1, Tn9) or inverted (most bacterial transposon elements) (2-4). We report here that the terminal regions of the Dictyostelium DIRS-1 element are inverted repeats of 330 base pairs (bp); all of the repeats on the right side of the transposon are extended by an additional 28 bp rich in A and T residues. We also show that the repeats are found interspersed in the genome, independent of the complete element. We discuss the possible origins of such genomic fragments.DIRS-1 hybridizes to a large number of differently sized cytoplasmic polyadenylylated RNAs that accumulate in a coordinated fashion during development (5). These RNAs appear to be mRNAs as they are polyadenylylated and are specifically associated with polysomes (unpublished data). Most of the RNAs carrying sequences complementary to DIRS-1 are induced in response to heat shock or to other "stresses" which precede the initiation of development (5). Moreover, one of the many genomic fragments related to DIRS-1 (clone pB41-6) contains a 15-base sequence that resembles a Drosophila heat shock promoter (5) and directs the synthesis of a heat-inducible RNA when introduced into yeast (6). We demonstrate here that the heat shock promoter responsible for the thermal induction of pB41-6 is contained within the terminal repeats of DIRS-1.

show abstract

“…The filters were prehybridized in a solution containing 5x SSC, 3x Denhardt solution, 0.2% SDS, and 50 pg of E. For the preparation of the RNA filters denatured poly(A+) RNA was fractionated on 1.5% agarose gels containing 6% formaldehyde as described by Rave et al (23). The RNA was then transferred to nitrocellulose paper as described by Mangiarotti et al (17). The filters were hybridized as described for the DNA dot-blot filters with denatured plasmid DNA that had been radioactively labeled by nick translation (106 Cerenkov cpm per ml of hybridization buffer; 8 x 106 Cerenkov cpm per ,ug of DNA) (19,24 8, and 9), 10 h (lanes 10, 11, and 12), or 15 h (lanes 13,14, and 15) after transfer to sporulation medium (SPO).…”

Section: Methodsmentioning

confidence: 99%

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

Percival‐Smith¹,

Segall²

1984

Mol. Cell. Biol.

103

106

View full text Add to dashboard Cite

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating aa cells and asporogenous aao cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the aa sporulation probe relative to the aa control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, aa, and aa cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified.

show abstract

Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition

Cited by 85 publications

References 18 publications

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Dictyostelium transposable element DIRS-1 has 350-base-pair inverted terminal repeats that contain a heat shock promoter.

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

Contact Info

Product

Resources

About