Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization.

Multiple nitrocellulose DNA filter replicas of plaques of in vitro generated recombinants of phage lambda and Saccharomyces cerevisiae have been screened by hybridization with 32P-labeled cDNA probes. These probes were representative of total poly(A)-containing RNA of yeast cells grown on acetate, galactose, glucose or maltose. This approach allows the use of specific differences in total RNA populations as probes for gene isolation. Five "galactose-induced" clones have been isolated. Expression of the RNA coding regions on at least two cloned sequences, Sc481 and Sc482, is regulated by genes known to control the expression of the structural genes required for the conversion of exogenous galactose to endogenous glucose-1-phosphate. One cloned sequence, Sc484, is expressed during growth on all carbon sources except glucose, and is not under control by the galactose regulatory genes. This clone contains a sequence that is repeated 3 times in the yeast genome. The cloned fragment Sc481 contains coding regions for all or part of three galactose"induced RNAs and may correspond to the GAL 1, GAL 7, GAL 10 gene cluster region of chromosome II.     

Isolation of a thiamine-binding protein from Saccharomyces cerevisiae.

Thiamine-binding protein was isolated from Saccharomyces cerevisiae by successive procedures of cold osmotic shock treatment, DEAE-cellulose chromatography and ultrafiltration. The purified thiamine-binding protein was an electrophoretically homogeneous molecule which appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. No thiamine-binding protein was observed by disc gel electrophoresis in the shock fluid released from yeast cells grown in the presence of 1 muM thiamine, indicating that the formation of this protein is regulated by exogenous thiamine as previously suggested.     

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Summary Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.     

Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences

Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.     

Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae.

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.     

A protein containing conserved RNA-recognition motifs is associated with ribosomal subunits in Saccharomyces cerevisiae.

Using PCR cloning techniques, we have isolated a Saccharomyces cerevisiae gene encoding a protein that contains two highly conserved RNA-recognition motifs. This gene, designated RNP1, encodes an acidic protein that is similar in sequence to a variety of previously isolated RNA binding proteins, including nucleolin, poly (A) binding protein, and small nuclear ribonucleoproteins. The RNP1 gene maps to the left arm of chromosome XIV centromere distal to SUF10. Haploid yeast containing a null allele of RNP1 are viable, indicating that RNP1 is dispensible for mitotic growth. However genomic Southern blot analysis indicated that several other loci in the S. cerevisiae genome appear to contain sequences similar to those in the RNP1 gene. The majority of the Rnp1 protein is cytoplasmic. Extra copies of RNP1 cause a decrease in levels of 80S monoribosomes. A fraction of Rnp1 protein cosediments on sucrose gradients with 40S and 60S ribosomal subunits and 80S monosomes, but not with polyribosomes.     

cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.     

G418 Selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae

The chromosomal delta sequences of the yeast Saccharomyces cerevisiae were employed as recombination sites to integrate the bacterial neo(r) gene and the yeast SUC2 gene into the yeast genome. A dominate selection method employing the aminoglycoside antibiotic G418 was used. Transformation efficiencies and growth behaviors of the transformants were studied. Transformants were obtained with more than 40 integrations; the majority of insertions were tandem with a maximum of three different insertion sites utilized at one time. After 70-100 generations of growth in nonselective medium, the high copy number SUC2-neo(r) integrants were found to be unstable; only minor instability was observed for the neo(r) and low copy number SUC2-neo(r) integrants. (c) 1996 John Wiley & Sons, Inc.