Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus.

Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeIII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains approximately 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.     

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).     

Kindred DNA amplification from two distinct populations of cDNA fragments

Kindred DNA amplification is a novel and cost-effective method developed to isolate common cDNA fragments between two distinct cDNA populations. Unlike subtractive hybridization, which discards common sequences, kindred DNA amplification isolates and amplifies these sequences within a single hybridization procedure. The utility of this method is demonstrated by cloning the genes in common between two different but metabolically homologous muscles, murine ventricular myocardium and soleus. The reliability of kindred DNA amplification was confirmed by Southern hybridization.     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

Mechanics and Dynamics of X-Chromosome Pairing at X Inactivation

At the onset of X-chromosome inactivation, the vital process whereby female mammalian cells equalize X products with respect to males, the X chromosomes are colocalized along their Xic (X-inactivation center) regions. The mechanism inducing recognition and pairing of the X's remains, though, elusive. Starting from recent discoveries on the molecular factors and on the DNA sequences (the so-called “pairing sites”) involved, we dissect the mechanical basis of Xic colocalization by using a statistical physics model. We show that soluble DNA-specific binding molecules, such as those experimentally identified, can be indeed sufficient to induce the spontaneous colocalization of the homologous chromosomes but only when their concentration, or chemical affinity, rises above a threshold value as a consequence of a thermodynamic phase transition. We derive the likelihood of pairing and its probability distribution. Chromosome dynamics has two stages: an initial independent Brownian diffusion followed, after a characteristic time scale, by recognition and pairing. Finally, we investigate the effects of DNA deletion/insertions in the region of pairing sites and compare model predictions to available experimental data.     

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.     

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.     

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.     

DNA序列信息的一种新的测度

根据信息理论给出了测度ＤＮＡ序列信息的一种新的方法,获得ＤＮＡ序列４个层次的信息量测度：Ｉｂ,Ｉｆ（１）,Ｉｆ（２）ａｎｄＩｆ（３）,这４种信息测度可分别用来测度ＤＮＡ的碱基序列、密码子序列、编码蛋白质序列和功能蛋白质序列的信息量。从Ｍ．ｅｄｕｌｉｓ的线粒体基因组中两个较短的编码蛋白质的ＤＮＡ序列和使用具有不同倍性的间并密码子组组成的模拟ＤＮＡ序列中所获得计算结果表明,这些信息测度确实能用来揭示所     

Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo. This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday     

Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. II. The structure of variant 1103 and its comparison to variant CVPS/1P2 (EcoRI res).

In an effort to characterize sites of recombination between SV40 and monkey DNA, we have determined the primary sequence of a large portion of the SV40 variant, designated 1103. This virus contains DNA sequences derived both from the wild type SV40 genome and from the permissive monkey cell in which the virus was propagated. Further, the monkey sequences included in the defective genome are homologous to both highly repeated monkey DNA (alpha component) and sequences that are infrequently repeated in the monkey genome. The regions of the 1103 genome where DNA sequences were determined include 1) the segments of the variant that surround joints connecting SV40 and monkey sequences, 2) the segment that contains the joint between monkey sequences of high and low reiteration frequency, and 3) the DNA segment of the variant that is homologous to monkey alpha component DNA. Comparison of the data obtained from the sequences analysis of the SV40 variants 1103 and CVP8/1/P2 (EcoRI res) (described in Wakamiya, T., McCutchan, T., Rosenberg, M., and Singer, M. (1979) J. Biol. Chem 254, 3584-3591) reveals certain similarities between the two that may be involved in eukaryotic recombination and defective variant formation.     

Two tuf genes in the cyanobacterium Spirulina platensis.

Probes derived from the tufA gene of Escherichia coli have been utilized to detect homologous sequences on Spirulina platensis DNA. A 6-kilobase-pair fragment of S. platensis DNA appears to contain two sequences homologous to the E. coli gene. Thus, as reported for gram-negative bacteria, the cyanobacterium presumably contains two tuf genes.     

The Search for Homology Does Not Limit the Rate of Extrachromosomal Homologous Recombination in Mammalian Cells

Mouse LTK(-) cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.     

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.     

Multiple mechanisms generate extrachromosomal circular DNA in Chinese hamster ovary cells.

Seven cloned small circular DNA molecules from CHO cells were sequenced and examined for the presence of homologies to each other and to a number of other functional sequences present in transposable elements, retroviruses, mammalian repeat sequences, and introns. The sequences of the CHO cell circular DNA molecules did not reveal common structural features that could explain their presence in the circular DNA population. A gene bank was constructed for CHO chromosomal DNA and sequences homologous to two of the seven small circular DNA molecules were isolated and sequenced. The nucleotide sequences present at the junction of circular and chromosomal DNA suggest that a recombination process involving homologous pairing may have been involved in the generation of one, but not the other, of the two circular DNA molecules.