Structure of the rhsA locus from Escherichia coli K-12 and comparison of rhsA with other members of the rhs multigene family.

The complete nucleotide sequence of the rhsA locus and selected portions of other members of the rhs multigene family of Escherichia coli K-12 have been determined. A definition of the limits of the rhsA and rhsC loci was established by comparing sequences from E. coli K-12 with sequences from an independent E. coli isolate whose DNA contains no homology to the rhs core. This comparison showed that rhsA comprises 8,249 base pairs (bp) in strain K-12 and that the Rhs0 strain, instead, contains an unrelated 32-bp sequence. Similarly, the K-12 rhsC locus is 9.6 kilobases in length and a 10-bp sequence resides at its location in the Rhs0 strain. The rhsA core, the highly conserved portion shared by all rhs loci, comprises a single open reading frame (ORF) 3,714 bp in length. The nucleotide sequence of the core ORF predicts an extremely hydrophilic 141-kilodalton peptide containing 28 repeats of a motif whose consensus is GxxxRYxYDxxGRL(I or T). One of the most novel aspects of the rhs family is the extension of the core ORF into the divergent adjacent region. Core extensions of rhsA, rhsB, rhsC, and rhsD add 139, 173, 159, and 177 codons to the carboxy termini of the respective core ORFs. For rhsA, the extended core protein would have a molecular mass of 156 kilodaltons. Core extensions of rhsB and rhsD are related, exhibiting 50.3% conservation of the predicted amino acid sequence. However, comparison of the core extensions of rhsA and rhsC at both the nucleotide and the predicted amino acid level reveals that each is highly divergent from the other three rhs loci. The highly divergent portion of the core extension is joined to the highly conserved core by a nine-codon segment of intermediate conservation. The rhsA and rhsC loci both contain partial repetitions of the core downstream from their primary cores. The question of whether the rhs loci should be considered accessory genetic elements is discussed but not resolved.     

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

Molecular and genetic studies on the euchromatin-heterochromatin transition region of the X chromosome of Drosophila melanogaster

A recombinant Charon 4 bacteriophage has been isolated on the basis of RNAs which are enriched in the head of the adult Drosophila melanogaster and hence are likely to be of neural origin. The cloned insert maps to the near vicinity of the uncoordinated locus in polytene chromosome band 19E8. This band is within the transition zone between the euchromatic and heterochromatic regions of the X chromosome, a region which has been well characterized cytogenetically. The insert contains both repetitious and low copy number sequences, some of which vary extensively in both frequency and restriction fragment size between different laboratory strains. One particular family of moderately repeated sequences occurs predominantly in divisions 19 and 20 of the X chromosome and perhaps the distally located X heterochromatin. The molecular landscape surrounding the initial entry point contains many repeated sequences and is thus unlike those observed in most published chromosomal walks. The possible significance of the presence of repeated sequence families in the distinct properties of this region are discussed.     

Absence of DNA sequence homology with genes of the Escherichia coli hemB locus in Shiga-toxin producing E. coli (STEC) O157 strains

By molecular cloning of chromosomal DNA of a human faecal Escherichia coli O6:non-motile strain, we identified a 1350-bp DNA segment which is commonly present in laboratory and wild-type E. coli strains but had no homology to DNA of Shiga-toxin producing E. coli O157, O145 and enteropathogens E. coli O55 strains. The nucleotide sequence of the 1350-bp segment cloned on plasmid pEO67 was determined (GenBank accession number AF087670) and a 97.2% sequence homology was found to a region of the E. coli hemB locus with an unknown gene function. The introduction of pEO67 into an STEC O157:H- strain had a stimulating effect on the growth of the recipient strain which was most expressed when bacteria were grown in iron depleted M9 medium with hemin added as the exogenous iron source. This growth effect was not observed with E. coli K-12 carrying pEO67. We suggest that the cloned gene is involved in iron uptake of E. coli and that the alteration in this part of the hemB locus is clonally inherited in genetically closely related STEC O157 and O55 strains.     

The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans.

The AROM locus of A. nidulans, which governs five consecutive steps in pre-chorismate aromatic amino acid biosynthesis, has been cloned in a bacteriophage vector. The nucleotide sequence of the locus reveals a single, open reading-frame of 4,812 base-pairs, apparently without introns. An internal segment of the A. nidulans AROM sequence has extensive homology with the E. coli aroA gene that encodes the 5-enolpyruvylshikimate 3-phosphate synthase.     

Nucleotide sequence of the asnA gene coding for asparagine synthetase of E. coli K-12.

We have subcloned the asnA gene of E. coli K-12, a gene coding for asparagine synthetase, from a previously cloned 6 mega-dalton segment of E. coli chromosome containing the DNA replication origin, ori, and asnA. The complete nucleotide sequence of the asnA gene was determined: the region of the structural gene extends 990 base-pairs at nucleotide positions 1434-2423 (see Fig. 3), which codes for a polypeptide of 330 amino-acid residues with a molecular weight of 36,688 daltons. The nucleotide sequences of the promoter and the ribosome-binding site of the gene are also assigned. We discuss the properties of its polypeptide.     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

A new locus, pilE, required for the binding of type 1 piliated Escherichia coli to erhythrocytes

Abstract We have identified a new locus required for mediating the binding of type 1 piliated Escherichia coli cells to guinea pig erythrocytes. This locus, pilE , was discovered after restrition site mutagenesis of a cloned segment of DNA containing the chromosomal pil region from a clinical strain of E. coli. pilE Mutants failed to agglutinate guinea pig erythrocytes but expressed pili were morphologically and antigenically indistinguishable from the parental ( pilE ⁺) strain. Construction of a chromosomal pilE mutation in E. coli K-12 was accomplished by introducing a restriction fragment containing a pilE lesion into the chromosome of a recBC sbcB host strain. Mutations in pilE could be complimented in trans by the addition of a cloned segment of DNA containing the parental pilE locus. Lesions in any of the genes required for pilus assembly also produced a hemagglutination minus phenotype suggesting that both the product(s) specified by the pilE locus and pili were required for hemagglutination. Hemagglutination experiments using partially purified pili also supported this suggestion.     

Structure and expression of cloned murine IFN-alpha genes

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.     

Y chromosomal DNA of Drosophila hydei

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.     

A human dihydrofolate reductase intronless pseudogene with an Alu repetitive sequence: multiple DNA insertions at a single chromosomal site

A dihydrofolate reductase (DHFR) pseudogene, hDHFR-psi 3 has been isolated from a human genomic DNA fragment library. Sequence analysis of this gene revealed a lack of introns and the presence of a tract of nine adenines, 90 bp downstream from the end of the coding sequence. These features suggest that hDHFR-psi 3 was derived from a processed RNA molecule that has been converted into DNA and inserted into a chromosome, analogous to the origin of three intronless human DHFR genes previously described. An interesting feature of hDHFR-psi 3 is the presence of a member of the Alu moderately repetitive DNA sequence family within the DHFR coding region. This Alu element is flanked by a 16 bp directly repeated DNA segment derived from DHFR coding sequences. The Alu element apparently has been inserted into the intronless DHFR pseudogene and thus, there have been two insertions at a single chromosomal locus. The hDHFR-psi 3 contains only the 3' half of the DHFR coding sequence. Immediately upstream from the directly repeated sequence before the Alu element is an adenine-rich tract. The DNA farther upstream is moderately repetitive and is related to neither DHFR nor Alu DNA sequence. Therefore, it seems possible that a third insertion has occurred at the same site further disrupting the hDHFR coding sequences.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Sequence variation in dispersed repetitive sequences in Saccharomyces cerevisiae

Two new dispersed repetitive DNA sequences related to the transposable element Tyl have been isolated from the genome of Saccharomyces cerevisiae. One sequence, designated Tyl-17, is present at about six copies per haploid genome, and one copy is located approximately 1000 base-pairs from the LEU2 locus on chromosome III. Tyl-17 is about the same size as Tyl (Cameron et al., 1979) and is flanked by δ sequences, but differs from Tyl by the presence of two large substitutions representing about 50% of the sequence. Tyl and Tyl-17 are found in a ‘head-to-head’ array in at least one cloned region of the yeast genome. Another sequence, designated Tyl-161, is situated about 9000 base-pairs from the PGK locus of chromosome III, and is structurally identical to Tyl except for the presence of a 1200 base-pair insertion near one end of the sequence element.     

Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens.

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.