A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

Meiosis and fertility of F1 hybrids between hexaploid bread wheat and decaploid tall wheatgrass (Thinopyrum ponticum)

As the first step in the transfer of barely yellow dwarf virus resistance and salt tolerance from decaploid tall wheatgrass (Thinopyrum ponticum) into hexaploid bread wheat (Triticum aestivum L.), octoploid intergeneric hybrids (2n = 8x = 56) were synthesized by crossing the tall wheatgrass cultivar Alkar with wheat cvs. Fukuhokomugi (Fuko) and Chinese Spring. (Fuko x Alkar) F₁ hybrids were studied in detail. The F₁ hybrids were perennial and generally resembled the male wheatgrass parent with regard to morphological features and gliadin profile. Most hybrids were euploid with 56 chromosomes and showed high chromosome pairing. On an average, in 6 hybrids 83.6% of the complement showed chiasmatic association, some between wheat and wheatgrass chromosomes. Such a high homoeologous pairing would be obtained if Ph1, the major homoeologous pairing suppressor in wheat, was somehow inactivated. Some of the Fuko x Alkar hybrids had high pollen fertility (18.5–42.0% with a mean of 31.5%) and high seed fertility (3–29 seeds wtih a mean of 12.3 seeds per spike), offering excellent opportunities for their direct backcrossing onto the wheat parent.     

Chromosome structure of Triticum longissimum relative to wheat

Homoeologous pairing at meiotic metaphase I was analyzed in T. longissimum x T. aestivum hybrids in order to reconfirm the homoeologous relationships of T. longissimum chromosomes to wheat. Hybrids between T. longissimum and Chinese Spring carrying the Ph1 gene or theph1b mutation, which showed low and high pairing levels, respectively, were used. Chromosome arms associated at metaphase I were identified by C-banding. The homoeology of chromosomes 1S ^l , 2S ^l , 3S ^l , 5S ^l and 6S ^l to wheat group 1,2, 3, 5, and 6 chromosomes, respectively, was confirmed. Chromsome arms 4S ^l S and 7S ^l S showed normal homoeologous relationships to wheat. The 4S ^l L arm carries a translocated segment from 7S ^l L relative to wheat. The 7S ^l L arm seldom paired, likely because this arm lost a relatively long segment and received a very short segment in the interchange with 4S ^l L. Available data suggest that translocation 4S ^l L/7S ^l L arose in the evolution of T. longissimum, which implies that this species was not the donor of the B genome of wheat.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

DOES RECOMBINATION CONSTRAIN NEUTRAL DIVERGENCE AMONG BACTERIAL TAXA?

A coalescence model for predicting the fate of neutral divergence among closely related taxa distinguishable as separate DNA sequence clusters is presented here. The model simulates iteratively the positive feedback between sequence divergence and sexual isolation among taxa, where increases in sequence divergence result in reduced recombination, and reduced recombination results in increased sequence divergence. Iteration of this feedback is continued until sequence divergence either converges on a steady state or reaches a runaway process. The eventual outcome of sequence divergence was shown to depend on four estimable population-genetic parameters: the expected intrataxon sequence diversity, the baseline rate of intertaxon recombination, the sensitivity of the recombination rate to sequence divergence, and the neutral mutation rate. The model can be used to determine whether neutral divergence among actual taxa is destined to stop at an equilibrium level, or whether neutral divergence will reach a runaway process. Application of the model to the group of taxa containing Bacillus subtilis and its closest relatives showed these taxa to be on a trajectory of unbounded neutral divergence from one another.     

Nucleotide polymorphism in the chalcone synthase-A locus and evolution of the chalcone synthase multigene family of common morning glory Ipomoea purpurea

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS-A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15-carbon three-ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS-A alleles from Mexican and American populations of common morning glory. American populations of this annual self-compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south-eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre-Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS-A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.