Ordered Clone Bank Research Articles

Cloning and nucleotide sequencing of the genes, rpIU and rpmA, for ribosomal proteins L21 and L27 of Escherichia coli.

The rpIU and rpmA genes that encode ribosomal proteins (r-proteins) L21 and L27 of Escherichia coli K-12 have been isolated from the ordered clone bank of this bacterium. They were found to be located at coordinates 3,351.7-3,352.3 kb on the physical map of E. coli. The nucleotide sequence of the cloned genes and their flanking regions indicated that the two r-protein genes compose an operon. Upstream of the two genes there is an open reading frame (ORF) in the opposite direction. The deduced polypeptide encoded by this ORF has a molecular weight of 35,215 and shows a significant degree of sequence similarity to the enzyme that is involved in the carotenoid biosynthesis and encoded by the crtE gene of carotenogenic bacteria and to prenyltransferases found in various organisms.

An ordered clone bank for chromosome I of Saccharomyces cerevisiae

Chromosome I of Saccharomyces cerevisiae DC5 rho 0 was dissected into segments with an average size of 14.0 kb and cloned into lambda phage vectors. The physical maps of the resultant clones, totaling 205.9 kb, were used to construct an ordered clone bank of this chromosome.

Correlation of a subset of the pLC plasmids to the physical map of Escherichia coli K-12.

We determined map positions of the Escherichia coli K-12 portions of a subset of the hybrid E. coli-ColE1 plasmids constructed by Clarke and Carbon. The probe DNA of pLC plasmids was labeled with digoxigenine-dUTP, hybridized to the 476 phage clones of the E. coli ordered clone bank miniset, which was adsorbed on a strip of nylon membrane filters, and detected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed color reaction. The total number of Clarke-Carbon plasmids we analyzed was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed with those reported previously, and the remaining 41 plasmids gave map positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it is presented, as well as a table which correlates the pLC plasmids with the clones of the E. coli ordered clone bank miniset on the basis of the hybridization data. We estimate that approximately one-half of the entire genome of E. coli was covered by the pLC plasmids used in this study.

Physical dissection and characterization of chromosomes V and VIII of Saccharomyces cerevisiae.

Chromosomes V and VIII of S. cerevisiae were dissected and ordered clone banks were constructed and characterized. Each bank contains almost the entire chromosome from the left to the right telomere except for a small gap in each case. The size of the banks constructed is in good agreement with the physical length of these chromosomes, 580 kb, estimated by pulsed-field gel electrophoresis. The remaining gap in the ordered clone bank of chromosome V was found to be only 1.6 kb in length and to contain a 1.5 kb-long portion of one of the two Ty elements located in tandem. The gap in the bank of chromosome VIII was 6.4 kb in length and contained four copies of the CUP1 gene. A genomic restriction map analysis of the corresponding region of chromosome VIII revealed that a unit of about 2 kb in length harbouring the CUP1 gene was repeated ten times in strain DC5 rho degrees which was used for the bank construction. A 588.5 kb-long high resolution physical map for chromosome V and a 585.6 kb-long one for chromosome VIII have thus been established.

Correlation of a subset of the pLC plasmids to the physical map of Escherichia coli K-12.

We determined map positions of the Escherichia coli K-12 portions of a subset of the hybrid E. coli-ColE1 plasmids constructed by Clarke and Carbon. The probe DNA of pLC plasmids was labeled with digoxigenine-dUTP, hybridized to the 476 phage clones of the E. coli ordered clone bank miniset, which was adsorbed on a strip of nylon membrane filters, and detected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed color reaction. The total number of Clarke-Carbon plasmids we analyzed was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed with those reported previously, and the remaining 41 plasmids gave map positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it is presented, as well as a table which correlates the pLC plasmids with the clones of the E. coli ordered clone bank miniset on the basis of the hybridization data. We estimate that approximately one-half of the entire genome of E. coli was covered by the pLC plasmids used in this study.

Cloning, characterization, and physical location of the rplY gene which encodes ribosomal protein L25 in Escherichia coli K12

The rplY gene of Escherichia coli K12 encoding ribosomal protein L25 was cloned from the ordered clone bank and located at coordinate 2,291 kb on the physical map of E. coli. Determination of the nucleotide sequence indicated that the coding region contains 285 nucleotide pairs including a translational initiator and terminator. The amino acid sequence of the protein deduced from the nucleotide sequence matched completely the sequence determined for ribosomal protein L25. The coding region was found to be preceded by a typical promoter-like sequence and was followed by a DNA region capable of forming a secondary structure characteristic of a transcriptional terminator. Thus, the gene was concluded to constitute a transcriptional unit (operon). A preliminary analysis by Northern blot supported this conclusion. The codon usage pattern of the rplY gene is characteristic of the ribosomal protein genes in E. coli.

Construction of an ordered clone bank and systematic analysis of the whole transcripts of chromosome VI ofSaccharomyces cerevisiae

By comparing sequences of restriction enzyme cleavage sites and their distance data, we sorted 384 lambda phage clones containing segments of chromosome VI of S. cerevisiae and constructed an ordered clone bank for this chromosome. The physical length of this bank is 269.7 kb. The bank contains the entire chromosome including the left telomere, but it is not certain whether it contains the right telomere as well. To estimate the number of genes present on this chromosome, we performed a series of Northern hybridization experiments using 157 restriction enzyme fragments prepared from the bank as hybridization probes and total poly(A)+ RNA from vegetatively growing cells. Thus, 97 distinct transcripts were identified. The relative abundance levels of individual transcripts were measured by comparing their band intensity with that of the RPO41 transcript. It was found that the transcripts from the genes located in the telomeric and centromeric regions are less abundant as compared to those from the genes in the central regions of both arms.

Chromosome III of Saccharomyces cerevisiae: An ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of 160 genes

Using lambda phage vector EMBL4, we isolated 344 clones containing segments of chromosome III of Saccharomyces cerevisiae, analysed their physical structure with eight restriction enzymes and sorted the data in contiguous groups with computer programmes. Furthermore, we performed Southern hybridizations between the sorted contiguous clone groups and interrelated them into larger groups. In this way, we constructed an ordered clone bank that covers almost the whole of chromosome III with a single gap of several kilobases in length. The consensus physical map thus obtained totals 334.6 kb, which is in good agreement with the size of this chromosome estimated by pulsed-field gel electrophoresis. Southern hybridization analysis with the DNA probes containing telomere-specific sequences showed that the bank contained a telomere at a position corresponding to the right arm terminus of chromosome III. Also, five Ty elements were found to be present. To estimate the number of genes on this chromosome and to analyse their levels of expression, we performed a series of Northern hybridization experiments using total poly(A)+ RNA from vegetatively growing cells and appropriate restriction enzyme fragments from the bank. Thus, we identified a total of 156 transcripts on chromosome III, indicating, on an average, one gene in every 2 kb on this chromosome. The transcripts were visually categorized into five groups according to their apparent levels of expression. It was found that the genes located near both termini are expressed only at low levels and that highly expressed genes are rather scattered over the chromosome.

Cloning and analysis of an Escherichia coli operon containing the rpmF gene for ribosomal protein L32 and the gene for a 30-kilodalton protein

The chromosomal DNA fragments of Escherichia coli K-12 were cloned into a mini-F cosmid, pRE435, after partial digestion with restriction endonuclease Sau3AI. The clones were first screened for PyrC+ and then for other genes, including rpmF encoding ribosomal protein L32 that had been mapped near pyrC (I. Janda, M. Kitakawa, and K. Isono, Mol. Gen. Genet. 201:443-436, 1985). Thus, we obtained a total of five rpmF-containing clones. The rpmF gene was located on the chromosomal segment in one of the clones (pAY2-5) by insertional mutagenesis with transposon gamma delta, followed by analysis of the gene products by the maxicell method. Hybridization analysis of clone pAY2-5 with the ordered clone bank (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) indicates that a gap at the 1,510-kilobase coordinates in the physical map of E. coli can be bridged by this clone. The nucleotide sequence of the region containing rpmF was accordingly established. In addition, the RNA transcripts from the chromosomal region containing rpmF were analyzed, and the transcriptional initiation sites were determined. The results suggest that rpmF forms an operon with the gene termed g30k which codes for a 30-kilodalton protein of unknown function. At least four transcripts were found to code for ribosomal protein L32.

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.

An ordered clone bank of E. coli genome: Toward understanding of the complete genetic architecture.

We have developed a new strategy for rapid restriction analysis and sorting of a large genomic library. Using this strategy, we have established a set of mutually overlapping lambda phage clones and the restriction map for eight six-base-recognizing enzymes that cover the whole of the 4700kb long genome of E. coli.

The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA.

The proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa is an extremely hydrophobic protein of 81 amino acid residues, which is imported into mitochondria as a precursor of mol. wt. 15 000. The primary structure of the imported form has now been determined by isolating and analyzing cDNA clones of the preproteolipid mRNA. An initial cDNA clone was identified by hybridizing total polyadenylated RNA to pooled cDNA recombinant plasmids from an ordered clone bank and subsequent cell-free translation of hybridization-selected mRNA. Further preproteolipid clones were identified at a frequency of 0.2% by colony filter hybridization. One isolated cDNA represented the major part of the preproteolipid mRNA. The nucleotide sequence showed 243 bases corresponding to the mature proteolipid and, in addition, 178 bases coding for an amino-terminal presequence . Non-coding sequences of 48 bases at the 5' end and of 358 bases at the 3' end plus a poly(A) tail were determined. The long presequence of 66 amino acids is very polar, in contrast to the lipophilic mature proteolipid, and includes 12 basic and no acidic side chains. It is suggested that the presequence is specifically designed to solubilize the proteolipid for post-translational import into the mitochondria.