Genetic Recombination

Mouse embryos lacking the retinoic acid receptor gene RXR(alpha) die in midgestation from hypoplastic development of the myocardium of the ventricular chambers and consequent cardiac failure. In this study, we address the issue of whether the RXRalpha gene is required in the cardiomyocyte lineage by generating mice that harbor a ventricular restricted deficiency in RXRalpha at the earliest stages of ventricular chamber specification. We first created a conditional ('floxed') allele of RXRalpha by flanking a required exon of the gene with loxP recombination sequences. To achieve ventricular myocardium-specific gene targeting, and to avoid potential transgenic artifacts, we employed a knock-in strategy to place cre recombinase coding sequences into the myosin light chain 2v (MLC2v) genomic locus, a gene which in the heart is expressed exclusively in ventricular cardiomyocytes at the earliest stages of ventricular specification. Crossing the MLC2v-cre allele with the floxed RXR...

Genetic recombination between viral genomes has been shown to contribute to the generation of genetic diversity during retrovirus infections. The role of recombination in the development of human immunodeficiency virus type 1 (HIV-1) zidovudine resistance was investigated as a possible cause of the formation of the linked Leu-41/Tyr-215 resistance genotype. Zidovudine resistance is conferred by the presence of subsets of four or five amino acid substitutions in the HIV-1 reverse transcriptase. Zidovudine therapy of asymptomatic HIV-1-infected individuals results in the selection of drug-resistant variants that posses defined combinations of the five zidovudine resistance mutations. The linked Leu-41/Tyr-215 resistance genotype appears central to the continued development of high-level zidovudine resistance. By using genetically tagged mutant viruses, it was possible readily to select recombinant viruses from mixed infections of Leu-41 and Tyr-215 single mutants in the presence of zi...

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they...

The present review summarizes the current state of knowledge about the genetics of pain-related phenomena and illustrates the scope and power of genetic approaches to the study of pain. We focus on work performed in our laboratories in Jastrzebiec, Poland; Portland, OR; and Los Angeles, which we feel demonstrates the continuing usefulness of classical genetic approaches, especially when used in combination with newly available molecular genetic techniques.

MOTIVATION: Several methods in molecular population genetics have recently been described to estimate the amount and pattern of the DNA polymorphism in natural populations, and also to test the neutral theory of molecular evolution. These methods are essential for ...

ABSTRACT Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.

Citrus black spot (Phyllosticta citricarpa) causes fruit blemishes and premature fruit drop, resulting in significant economic losses in citrus growing areas with summer rainfall across the globe. The mating type locus of P. citricarpa has recently been characterized, revealing the heterothallic nature of this pathogen. However, insight into the occurrence of mating and the impact of completing the sexual cycle of P. citricarpa was lacking. To investigate the occurrence and impact of sexual reproduction, we developed a method to reliably, and for the first time, produce ascospores of P. citricarpa on culture media. To demonstrate meiosis during the mating process, we identified recombinant genotypes through multilocus genotyping of single ascospores. Because the process of fertilization was not well understood, we experimentally determined that fertilization of P. citricarpa occurs via spermatization. Our results demonstrate that P. citricarpa is heterothallic and requires isolates ...

We have complemented the riboB2 mutation of Aspergillus nidulans by transformation with a plasmid library of wild-type (wt) sequences. We have isolated, by marker rescue from a riboB+ transformant, a plasmid that complements riboB2 efficiently. From this plasmid we have subcloned an A. nidulans sequence that complements riboB2 efficiently and that integrates by homologous recombination at a site closely linked to the riboB locus. We conclude that this sequence contains the wt riboB+ allele.

An efficient freeze-drying cycle for recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formulations, which contained glycine and sucrose as excipients, was developed. Development was based on characterizing the frozen formulations by thermal analysis and by examining the effect of various lyophilization process parameters on the sublimation rate of ice. Thermal analysis showed that the metastable glass of glycine in frozen formulation could be devitrified by slowly warming the frozen product to -15 degrees C. During drying, the sublimation rate of ice was increased as a linear function of the difference between the vapor pressure of ice at the product temperature (PO) and the chamber pressure (PC). Therefore, the product temperature (Tp) was maintained as high as possible at temperatures below Tg' of the formulation, in order to maximize the PO without allowing the collapse of cake. Although various combinations of shelf temperatures and chamber pressures could be u...

Two plasmids were constructed consisting of the E. coli vector pACYC184 and the cyanobacterial plasmid pUC1. These recombinants, designated pUC104 and pUC105, can be transformed to E. coli K12 as well as to the cyanobacterium Anacystis nidulans R2 and in both hosts they express their antibiotic markers. pUC104 and pUC105 differ with respect to the location and the orientation of the pACYC184 segment in pUC1. pUC104 was found to be stable under all circumstances. Transformation of pUC105 to A. nidulans R2 gave intact plasmids when chloramphenicol was the selective agent, but upon ampicillin selection a deletion derivative was produced identical to pUC1. Further characteristics of pUC104 and pUC105 are described and their usefulness as cloning vectors is discussed.

G: enetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the r...

Gene targeting in plants through homologous recombination has been sparsely reported, although notable breakthroughs have been achieved in recent years. In particular, the use of zinc finger nucleases to promote homologous end joining has revived the promise that homologous gene targeting could someday become practical for plant genetic engineering. An alternative and complementary approach that has progressed steadily over the years has been recombinase-mediated site-specific integration. In this approach, a first recombination site is introduced into the genome to serve as a target for inserting subsequent DNA. Here, we describe the method for generating the chromosomal target and the subsequent insertion of new DNA into the chromosomal target by Bxb1-mediated site-specific integration. This method would permit the comparison of different molecular constructs at the same genomic locations.

We have identified 12 individuals who are heterozygous for a chromosome with three a-globin genes. We determined the presence of the third a-globin locusby restric-tion endonuclease digestion and hybridization with a-globin cDNA probes. The three a-globin loci residedin ...