Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

Interruption of a human nuclear sequence homologous to mitochondrial DNA by a member of the KpnI 1.8 kb family.

We reported that several DNA sequences homologous to mitochondrial DNA (mtDNA) are present in the human nuclear genome (Tsuzuki et al. (1983) Gene 25, 223-229). Detailed Southern blot analyses revealed that one of such sequences is interrupted by a repetitive sequence about 1.8 kb long, and that the insert is one member of the dispersed repeated DNA sequences of the KpnI 1.8 kb family. Nucleotide sequence analysis showed that the KpnI 1.8 kb DNA is flanked with imperfect 15-base pair (bp) direct repeats of mtDNA. This KpnI 1.8 kb DNA has an A-rich sequence at its 3'-end, and has a considerable homology with one of the published cDNA sequences homologous to one of the human KpnI families and also to one of the African green monkey KpnI families, KpnI-LS1. These structural features suggest that the KpnI 1.8 kb DNA is a movable element and is inserted within the mtDNA-like sequence by an RNA-mediated process.     

Identification of a telomeric DNA sequence in Trypanosoma brucei

A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5' CCCTAA 3', was identified as being telomeric by several criteria. This sequence was specifically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3' ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence.     

Human DNA sequences isolated with an immunoglobulin switch region probe: sequence,chromosomal localization,and restriction fragment length polymorphisms

Summary We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias. Burkitt lyphhomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 40 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig indenpendent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination.     

Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.     

Possible insertion sequences in a mosaic genome organization upstream of the exotoxin A gene in Pseudomonas aeruginosa.

Nucleotide sequence and Southern hybridization data revealed a mosaic genome organization in a region that extends several thousand base pairs upstream of the exotoxin A (toxA) gene in Pseudomonas aeruginosa. An interstrain comparison of DNA in this region showed a pattern of alternating segments of homologous and nonhomologous sequences. Two nonhomologous elements, approximately 1 kilobase pair upstream of the gene in strains PA103 and Ps388, were characterized in more detail. The sequence elements, denoted IS-PA-1 and IS-PA-2 for the different strains, are about 1,000 and 785 base pairs long, respectively, and have 5-base-pair direct repeats at their boundaries, consistent with their being DNA insertion sequences. The distribution of these elements in 34 different strains was determined. IS-PA-1 was found in a single copy upstream of toxA in half of the strains and was found in two copies in four of the strains. Some strains contained neither element, and one strain carried both. The genome of another strain, WR5, which lacks toxA, was shown to contain a 350-base-pair region that was highly homologous to DNA sequences located just upstream of toxA in other strains. The WR5 genome lacked several kilobase pairs of DNA that was found both upstream and downstream of this homologous region in the other strains.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Avian myeloblastosis virus transforming gene is related to unique chicken DNA regions separated by at least one intervening sequence.

Identification of several additional restriction endonuclease sites within the cellular substitution (amv) inserted into the avian myeloblastosis virus proviral genome has permitted us to isolate different regions of the amv sequence. These subsets of the avian myeloblastosis virus transforming gene have been cloned in the plasmid pBR322 and used as hybridization probes to investigate the topology of homologous (proto-amv) normal chicken DNA sequences. The results showed that the cellular proto-amv sequences in C/O chicken DNA are interrupted by at least one intervening sequence. A partial arrangement of the proto-amv sequences is presented.     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Interaction of linear homologous DNA duplexes via Holliday junction formation.

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.     

Sequence-specific DNA uptake in transformation of Neisseria gonorrhoeae

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.     

N-terminal sequencing of low-molecular-mass components in cyanobacterial photosystem II core complex. Two components correspond to unidentified open reading frames of plant chloroplast DNA

We recently reported the presence of several low-molecular-mass protein components in the PS II O2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus [(1989) FEBS Lett. 244, 391-396]. Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing. There were two components in the 4.7 kDa region, both having a blocked N-terminus. One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts. The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II. The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.     

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Analysis of the chromocenter DNA composition in polytene chromosomes of Drosophila orena ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore 1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore 1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore 1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.     

Three-stranded DNA structure; is this the secret of DNA homologous recognition?

A novel type of triple-stranded DNA structure was proposed by several groups to play a crucial role in homologous recognition between single- and double-stranded DNA molecules. In this still putative structure a duplex DNA was proposed to co-ordinate a homologous single strand in its major groove side. In contrast to the well-characterized pyrimidine-purine-pyrimidine triplexes in which the two like strands are antiparallel and which are restricted to poly-pyrimidine-containing stretches, the homology-specific triplexes would have like strands in parallel orientation and would not be restricted to any particular sequence provided that there is a homology between interacting DNA molecules. For many years the stereo-chemical possibility of forming homology-dependent three- or four-stranded DNA structures during the pairing stage of recombination reactions was seriously considered in published papers. However, only recently has there been a marked increase in the number of papers that have directly tested the formation of triple-stranded DNA structures during the actual pairing stage of the recombination reaction. Unfortunately the results of these tests are not totally clear cut; while some laboratories presented experimental evidence consistent with the formation of triplexes, others studying the same or very similar systems offered alternative explanations. The aim of this review is to present the current state of the central question in the mechanism of homologous recombination, namely, what kind of DNA structure is responsible for DNA homologous recognition. Is it a novel triplex structure or just a classical duplex?     

The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA.

Low salt extracts of chicken oviduct nuclei contain a DNA binding protein with high affinity for specific DNA sequences in the flanking regions of the chicken lysozyme gene. Two of the three binding sites found within a total of 11 kb upstream from the promoter are located only 92 bp apart from each other. Upon comparison of the DNA binding sites, the symmetrical consensus sequence 5'- TGGCANNNTGCCA -3' can be deduced as the protein recognition site. This sequence is the central part of 23 to 25 base pairs protected by the DNA binding protein from DNAase I digestion. A homologous binding activity can be detected in nuclei from several chicken tissues and from mouse liver.     

A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes

Mouse genomic DNA contains multiple copies of sequences homologous to the Drosophila "Krüppel," a member of the "gap" class of developmental control genes of the fruit fly. The most interesting aspect of the homologous region is that, like Xenopus TFIIIA, it contains multiple finger-like folded domains capable of binding to nucleic acids. We have isolated six individual phages from a mouse genomic library on the basis of their DNA homology to Krüppel finger-coding probes, and describe here the DNA sequence and expression of two such clones containing finger-like structures. Upon differentiation of mouse teratocarcinoma cell line F9 with retinoic acid and cAMP, the expression of both genes was drastically reduced, and in one instance was undetectable. Each of the several other eukaryotic DNAs analyzed contained multiple copies of homologous genes with putative finger structures, indicating the presence of a finger-containing multigene family in higher organisms.