Bacteriophage lambda cloning vehicles for studies of genetic recombination

A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.     

A genetic and structural study of genome rearrangements mediated by high copy repeat Ty1 elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.     

Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification

Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells.     

携带污染物降解基因的可移动基因元件及其介导的生物修复

可移动基因元件(mobile genetic elements,MGEs)在环境微生物群落中的水平转移是细菌基因组进化和适应特定环境压力的重要机制.在污染土壤和水体中接种携带具有降解基因MGEs的菌株后,随着MGEs的水平基因转移,可使降解基因转移至具有竞争性的土著微生物中并在其中表达,从而不必考虑供体菌在环境中是否能够长期存活.这种由可移动降解基因元件水平转移介导的生物修复为探索新的生物修复途径提供了可行性.本文重点综述了环境样品中携带降解基因MGEs的多样性及其在促进污染物降解过程中的重要作用,介绍了从环境样品中分离代谢MGEs的方法,并列举了在污染土壤、活性污泥、其他生物反应器等生态系统中MGEs水平转移的几个实例.     

Major influence of repetitive elements on disease-associated copy number variants (CNVs)

Copy number variants (CNVs) are important contributors to the human pathogenic genetic diversity as demonstrated by a number of cases reported in the literature. The high homology between repetitive elements may guide genomic stability which will give rise to CNVs either by non-allelic homologous recombination (NAHR) or non-homologous end joining (NHEJ). Here, we present a short guide based on previously documented cases of disease-associated CNVs in order to provide a general view on the impact of repeated elements on the stability of the genomic sequence and consequently in the origin of the human pathogenic variome.     

Reproductive cloning and a (kind of) genetic fallacy

Many people now believe that human reproductive cloning--once sufficiently safe and effective--should be permitted on the grounds that it will allow the otherwise infertile to have children that are biologically closely related to them. However, though it is widely believed that the possession of a close genetic link to our children is morally significant and valuable, we argue that such a view is erroneous. Moreover, the claim that the genetic link is valuable is pernicious; it tends to give rise to highly undesirable consequences, and therefore should be combated rather than pandered to. The emphasis on the genetic is unwarranted and unfortunate; rather than giving us moral reason to support reproductive cloning in the case of infertility, the fact that cloning requests are likely to be motivated by the genetic argument gives us reason to oppose its availability.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning

The construction of two new derivatives of the bacteriophage cloning vector M13mp2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment of Escherichia coli beta-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of M13mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.     

DNA cloning demonstrates high genetic heterogeneity in populations of the subaerial green alga Trentepohlia (Trentepohliales,Chlorophyta)

Mats of the green alga Trentepohlia, a genus widely distributed in the tropics as well as temperate regions, have always been perceived as homogeneous (i.e., formed by only one species). As such, their general nature and specific feature play a supportive role in the species delimitation. However, the presence of morphologically dissimilar thalli was observed under the light microscope and when cultivating a piece of a single mat. To address this, we performed DNA cloning of the rbcL gene on mat fragments of T. abietina, T. annulata, T. jolithus and T. umbrina sampled in Europe to reveal if they are composed of one or more species. We revealed that more Trentepohlia haplotypes may coexist in a single mat. In consideration of this, we conclude that the use of material isolated in unialgal culture will be almost mandatory for a taxonomic reassessment of this complicated genus. Another direct implication of this problem is that herbarium specimens consisting of field‐collected material should not be used for direct sequencing. We further hypothesize the reasons why multiple haplotypes of Trentepohlia occur more frequently in the tuft‐like mats. Possibly, fragments and/or cells of other microalgae, including other species of Trentepohlia, might be retained in such mats more easily than in the crustose trentepohlialean mats.     

PSR (paternal sex ratio) chromosomes: the ultimate selfish genetic elements

PSR (paternal sex ratio) chromosomes are a type of supernumerary (or B) chromosomes that occur in haplodiploid arthropods. They are transmitted through sperm but then cause loss of the paternal chromosomes (except themselves) early in development. As a result, PSR chromosomes convert diploid fertilized eggs (which would normally develop into females) into haploid males that carry a PSR chromosome. Because they act by completely eliminating the haploid genome of their hosts, PSR chromosomes are the most extreme form of selfish or parasitic DNA known. PSR was originally described in the parasitic wasp Nasonia vitripennis (Pteromalidae). A second PSR chromosome has been found in Trichogramma kaykai, an egg parasitoid from a different family of Hymenoptera (Trichogrammatidae). We argue that PSR chromosomes are likely to be widespread in haplodiploid organisms, but have so far gone under reported due to a paucity of population genetic studies in haplodiploids. We describe the two known PSR systems and related phenomena, and models indicating the conditions conducive to increase of PSR like chromosomes in haplodiploids.     

Analysis of promoter mutations in the histidine transport operon of Salmonella typhimurium: use of hybrid M13 bacteriophages for cloning, transformation, and sequencing.

Mutations that cause an increased level of expression of the histidine transport operon were isolated and characterized genetically. Five independently isolated promoter-up mutations were transferred to an M13 hybrid phage that carries the histidine transport operon, and their nucleotide sequences were determined. For all five mutations, the change was the same as the one previously determined for promoter-up mutation dhuA1: a C-to-T change in the Pribnow box rendered this region more homologous to the consensus sequence. Methods for enabling Salmonella typhimurium to support growth of M13 phage effectively and for easy transfer of chromosomal mutations onto the hybrid phage are presented.     

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.     

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.     

Inversion of chromosome 2 (p11p13): Frequency and implications for genetic counselling

Summary We report a case of an X-autosome translocation t(X;4)(q13;p16) found in both sexes in three generations. The anomaly was diagnosed in a couple referred for cytogenetic investigation as a result of three spontaneous abortions. With the exception of the miscarriages there are no particularities in the gynecologic data of the woman or in the pedigree. In all 50 lymphocytes and in 66 of 68 fibroblasts investigated the normal X chromosome was the late replicating one.