Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.     

Disruption of the Pfg27 locus by homologous recombination leads to loss of the sexual phenotype in P. falciparum.

Transmission of malaria depends upon the differentiation and development of the sexual stages of the parasite. In Plasmodium falciparum, it is a complex, multistage process, involving the expression of a large number of sexual stage-specific proteins. Pfg27 is one such protein, abundantly expressed at the onset of gametocytogenesis. We report successful disruption of the Pfg27 locus using homologous recombination and show that it is essential for the maintenance of the sexual phenotype. Transfectants lacking Pfg27 abort early in sexual development, resulting in vacuolated, highly disarranged, and disintegrating parasites. This suggests a critical role for Pfg27 in the sexual development of the parasite.     

A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.     

Ac insertion site affects the frequency of transposon-induced homologous recombination at the maize p1 locus

The maize p1 gene regulates the production of a red pigment in the kernel pericarp, cob, and other maize floral tissues. Insertions of the transposable element Ac can induce recombination between two highly homologous 5.2-kb direct repeat sequences that flank the p1 gene-coding region. Here, we tested the effects of the Ac insertion site and orientation on the induction of recombination at the p1 locus. A collection of unique p1 gene alleles was used, which carry Ac insertions at different sites in and near the p1 locus, outside of the direct repeats, within the direct repeat sequences, and between the direct repeats, in both orientations. Recombination was scored by the numbers of colorless pericarp sectors (somatic frequency) and heritable mutations (germinal frequency). In both the somatic and germinal tests, the frequency of homologous recombination is significantly higher when Ac is inserted between the direct repeats than when Ac is inserted either within or outside the repeats. In contrast, Ac orientation had no significant effect on recombination frequency. We discuss these results in terms of the possible mechanisms of transposon-induced recombination.     

Sequence variation between alleles reveals two types of copy correction at the 27-kDa zein locus of maize

In many inbred lines of maize, two 27-kDa storage protein (zein) genes are found within tandem duplications of 12 kb. Both genes of the duplicated allele from the maize inbred line A188 were sequenced and compared to a similar duplicated allele in another inbred line, W22, and to a single-copy allele in the inbred line W64A. The comparisons reveal interesting patterns in the distribution of sequence changes between these alleles. Differences between the two duplicated alleles that are conserved between the two genes of each allele are found exclusively in the 5' region. In contrast, differences between the individual genes of each allele in the 3' region are conserved between the two alleles. The first case is indicative of an intraallelic copy correction mechanism, whereas the second may result from interallelic copy correction. These may be mediated by gene conversion processes, as previously described for other multigene families.     

3'-end processing of the maize 27 kDa zein mRNA

Cis -regulatory elements involved in the mRNA 3'-end processing of the 27 kDa zein gene have been investigated by deletion and site-directed mutagenesis analyses. In the 3' flanking region of the 27 kDa zein gene, several AATAAA-like sequences and a sequence resembling the mammalian GT-rich sequence are present around the polyadenylation sites. Among the multiple AATAAA-like sequences, the duplicated AATGAA motifs, located 30–40 bp upstream from the polyadenylation sites, have been shown to play roles as polyadenylation signals. Although either of the two AATGAA motifs can function as a polyadenylation signal in chimeric gene constructs, the one proximal to the polyadenylation sites is likely to be the functional polyadenylation signal in the 27 kDa zein gene. Deletion of the downstream GT-rich sequence as well as alteration of the sequence surrounding the poly-adenylation sites has little effect on the mRNA 3'-end processing. However, the sequence elements located upstream from the polyadenylation signals are essential for the mRNA 3'-end processing. Mutations in the AATGAA motifs or the upstream sequences reduced the level of a reporter gene expression. A model depicting the mechanism involved in the 3'-end processing of the 27 kDa zein mRNA is presented.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.     

Intrachromosomal homologous recombination in Arabidopsis induced by a maize transposon

In plants, the frequency of spontaneous intrachromosomal homologous recombination is low. Here, we show that a maize transposable element greatly stimulates intrachromosomal homologous recombination between direct repeat sequences in Arabidopsis. Plants were transformed with a construct (GU-Ds-US) containing a Ds (Dissociation) transposable element inserted between two partially deleted GUS reporter gene segments. Homologous recombination between the overlapping GUS fragments generates clonal sectors visible upon staining for GUS activity. Plants containing the GU-Ds-US construct and a source of Ac (Activator) transposase showed an over 1000-fold increase in the incidence of recombination relative to plants containing the same construct but lacking transposase. Transposon-induced recombination was observed in vegetative and floral organs, and several germinally transmitted events were recovered. Transposon-induced recombination appears to be a general phenomenon in plants, and thus may have contributed to genome evolution by inducing deletions between repeated sequences. Received: 28 September 1999 / Accepted: 19 November 1999     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

The uraA locus and homologous recombination in Mycobacterium bovis BCG.

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity. The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines.     

A new allele at the Pgd locus in pigs

A new electrophoretic variant of porcine 6-phosphogluconate dehydrogenase (PGD) is described. The new variant, PGD C, has been shown to be controlled by a third allele, PgdC, at the Pgd locus.     

Internode length in Pisum. A new allele at the Lh locus

A new allele at the Lh locus has been identified in Pisum sativum L. and named lhⁱ . This allele results in reduced GA levels in young shoots, and a dwarf phenotype. Gas chromatography-selected ion monitoring (GC-SIM) with dideuterated internal standards has been used to demonstrate a quantitative relationship between the level of endogenous GA₁ and internode length using the three alleles ( Lh, lh and lhⁱ ) at the Lh locus. These results are consistent with previous findings in peas (for alleles at the Le locus) and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis and support the role of GA₁ as the major native GA in peas with biological activity in its own right. However, in contrast to alleles at the Le locus, GA₂₀ levels are also reduced in lh and lhⁱ plants. The lhⁱ allele also has possible pleiotropic effects on seed abortion, leading to a reduction in seed yield compared to plants homozygous for the previously characterised Lh or lh alleles.