Correction of accelerated autoimmune disease by early replacement of the mutated lpr gene with the normal Fas apoptosis gene in the T cells of transgenic MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop a generalized autoimmune disease which includes increased autoantibody production, glomerulonephritis, and development of lymphadenopathy. The lpr genetic defect has been identified as a mutation in the Fas apoptosis gene that results in low expression of Fas mRNA. To determine the significance of the lpr mutation and T cells in the development of the autoimmune disease, we constructed transgenic MRL-lpr/lpr mice using a full-length murine Fas cDNA under the regulation of the T-cell-specific CD2 promoter and enhancer. Here we show that the early correction of the lpr gene defect in T cells eliminates glomerulonephritis and development of lymphadenopathy and decreases the levels of autoantibodies. In this model, early correction of the lpr defect in T cells is sufficient to eliminate the acceleration of autoimmune disease even in the presence of B cells and other cells that express the mutant lpr gene.     

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.     

Dominant male sterility in mice caused by insertion of a transgene.

While examining a series of transgenic mouse lines carrying the HCK protooncogene, we encountered one line in which males hemizygous for the transgene were sterile. The sterile males mated normally but failed to impregnate females. Light and electron microscopy revealed that spermatogenesis proceeds normally until nuclear condensation, which occurs but gives rise to a variety of abnormally shaped nuclei. Expression of the transgene was not detectable. Thus, the insertion itself probably caused the abnormal phenotype by disrupting a gene (or genes) important in spermatogenesis. The mutation is genetically dominant, causing an abnormal phenotype even though the sterile mice carry an ostensibly normal counterpart of the disrupted locus. The mutant phenotype is completely penetrant only in some genetic backgrounds, suggesting a modifying influence from a second locus. Junctions between the inserted transgene and adjoining cellular DNA were cloned, allowing us to confirm the heterozygous nature of the genetic disruption and to detect and associated deletion. We have designated the mutation Lvs (lacking vigorous sperm) and presume that it may define a previously undescribed locus important in spermatogenesis.     

De novo insertion of an intron into the mammalian sex determining gene, SRY

Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or “exon theory of genes,” proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or “insertional theory of introns,” proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron–exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene, SRY, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a de novo insertion of an intron into the SRY gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function.     

A transposable element from Halobacterium halobium which inactivates the bacteriorhodopsin gene.

We describe the characterization of a transposable element from an archaebacterium. The bacteriorhodopsin genes from the wild-type and two mutant Halobacterium halobium strains have been cloned as BamHI fragments in pBR322. The cloned DNA fragments from the two mutants both contain a 1.1-kilobase-pair insertion sequence (ISH1) near the NH2 terminus of the bacteriorhodopsin coding sequence. ISH1 is present in the two mutants in an identical palindromic site but in opposite orientations. The complete sequence of ISH1 has been determined; it is 1,118 nucleotides long, it has 8-base-pair interrupted inverted repeats at the ends, and it duplicates an 8-base-pair (A-G-T-T-A-T-T-G) target sequence upon insertion. As for most eukaryotic and some prokaryotic transposable elements, the sequence of the ISH1 begins with T-G and ends in C-A. ISH1 contains an open reading frame 810 nucleotides long and codes for an RNA approximately 900 nucleotides long. The copy number of ISH1 ranges from one to five or more in different H. halobium strains. In at least one of the strains, one copy of ISH1 is present also on a plasmid DNA.     

The maize transposable element system Ac/Ds as a mutagen in Arabidopsis: identification of an albino mutation induced by Ds insertion.

A two-component transposon system based on the Ac element of maize was used as a mutagen in Arabidopsis thaliana. Transposition of a Ds element marked with a hygromycin-resistance gene was activated from four different locations in the Arabidopsis genome. The progeny of 201 plants carrying independent transposition events were screened for mutants with severe, visible phenotypes. Seven mutants were identified and four of them were analyzed genetically. Three mutations were shown to be very closely linked to a transposed copy of the element. Moreover, a mutation (alb3) causing an albino phenotype was conclusively shown to be caused by insertion of the Ds element: somatic and germinal reversion of the mutation occurred in the presence of the transposase gene but not in its absence, and in three revertants the Ds had excised from its position in the mutant line. The DNA adjacent to Ds in the mutant was isolated and it was demonstrated that revertants retained part of the 8-bp duplication caused by insertion of Ds. These experiments indicate that the Ac/Ds system can be used as an insertional mutagen in the heterologous host Arabidopsis, which will permit the isolation of genes from this species by transposon tagging.     

黄芪对实验性病毒性心肌炎细胞凋亡及Fas/FasL基因转录的影响

目的　探讨黄芪对实验性病毒性心肌炎细胞凋亡及Fas/FasL基因的影响。方法　Balb/c小鼠 2 4只腹腔感染柯萨奇病毒B3(CVB3)后随机分成 2组 :治疗组 (每天腹腔注射黄芪注射液 10 0mg/ 10g)及对照组。第 8d取心脏标本 ,进行病理学检查、细胞凋亡TUNEL法检测以及逆转录聚合酶链反应 (RT PCR)分析Fas和FasLmRNA的表达水平。结果　①黄芪治疗组病变积分明显低于对照组 (P <0 0 1)。②黄芪治疗组心肌细胞凋亡指数 (AI)明显低于对照组 (P <0 0 1)。③黄芪治疗组心肌组织Fas和FasL的mRNA相对表达量均明显低于对照组 (P <0 0 1,P <0 0 5 )。结论　黄芪可通过下调病毒性心肌炎小鼠心肌组织Fas和FasL基因转录 ,减少心肌细胞凋亡和心肌损伤。     

Homologue destabilization by a putative transposable element in Drosophila melanogaster.

We postulate the presence of a transposable element, designated the L factor, to explain the properties of an unstable X chromosome and its derivatives. These chromosomes generate recessive lethal mutations at high rates, as does a stable X chromosome that has been associated with them for only one generation. The stable X chromosome does not become highly mutable in the absence of the unstable X chromosome, even when autosomes from the unstable stock are present. These facts suggest that the L factor is confined to the X chromosome and that it transposes to other X chromosomes paired with it. We propose the term "homologue destabilization" to denote the change in the stable chromosome brought about by this transposition. The lethal mutations caused by the L factor occur preferentially in the region around the cut wing locus (ct) and are sometimes associated with recognizable chromosome aberrations. The breakpoints of these aberrations are most often in the vicinity of ct, implying that the L factor is located near ct on the unstable chromosome, but it may reside at other sites as well. Alternately, the ct region may simply be a preferred target for the insertion of this transposable element.     

Arterial injuries caused by the insertion of an intra-aortic counterpulsion balloon

Insertion of an intra-aortic counterpulsion balloon was necessary following 283 surgical procedures with the heart pump at Hospital Broussais. One instance of vascular trauma at the site of insertion of the balloon was reported in 36 cases (13 p. cent). This was treated according to the severity of the arterial lesion with the following techniques: closure with Goretex venous patch (n = 25), short ilio-femoral by-pass (n = 9) and extra-anatomical femoro-femoral by-pass (n = 2). The overall mortality of the patients who required an intra-aortic balloon was 19 p. cent, including one case of retrograde aortic dissection at the site of insertion of the balloon. The morbidity related to the insertion of the balloon was as follows: one leg amputation, two disabling intermittent claudication episodes.     

Highly structured sequence homology between an insertion element and the gene in which it resides

The recessive allele for soybean seed lectin results from the insertion of a DNA segment (designated Tgm1) into the coding region of the gene. The termini of Tgm1 display structural features characteristic of a transposable element. The complete sequence of Tgm1 contains 3550 base pairs (bp) and can be divided into three regions (left arm, mid-section, and right arm). No large open reading frames were found, but an extensive, highly structured border with homology to the lectin gene was revealed. The left border (726 bp) comprising most of the left arm and extreme right border (144 bp) of the right arm consist of various forms of a basic 54-bp repeating unit. This 54-bp unit is comprised of a stem-loop structure and interhairpin sequence that occurs 13 times in the left arm and 2 times in the right arm of Tgm1. Progressively degenerate forms of this repeating unit appear toward the termini of Tgm1, but the dyad symmetry remains highly conserved. Seven nucleotides (A-C-A-T-C-G-G and its complement) maintained within the stem also appear as a subset of inverted repeats found at nearly equal distances from the target site in the lectin gene. Together with the inverted repeat termini and a duplication in the left arm, this 7-bp sequence occurs a total of 33 times in Tgm1. We infer that the dyad symmetries containing this sequence are involved in target gene selection. The repeating unit format of Tgm1 describes a distinct class of eukaryotic elements that includes representatives known to be mobile in snapdragon and maize.     

Abetalipoproteinemia caused by maternal isodisomy of chromosome 4q containing an intron 9 splice acceptor mutation in the microsomal triglyceride transfer protein gene.

Uniparental disomy (UPD), a rare inheritance of 2 copies of a single chromosome homolog or a region of a chromosome from one parent, can result in various autosomal recessive diseases. Abetalipoproteinemia (ABL) is a rare autosomal recessive deficiency of apoB-containing lipoproteins caused by a microsomal triglyceride transfer protein (MTP) deficiency. In this study, we describe a patient with ABL inherited as a homozygous intron 9 splice acceptor G(-1)-to-A mutation of the transfer protein gene. This mutation alters the splicing of the mRNA, resulting in a 36 amino acids, in-frame deletion of sequence encoded by exon 10. We analyzed chromosome 4, including MTP gene (4q22-24), using short tandem repeat markers. The proband has only his mother's genes in chromosome 4q spanning a 150-centimorgan region; ie, segmental maternal isodisomy 4q21-35, probably due to mitotic recombination. Nonpaternity between the proband and his father was excluded using 6 polymorphic markers from different chromosomes (paternity probability, 0.999). Maternal isodisomy (maternal UPD 4q) was the basis for homozygosity of the MTP gene mutation in this patient.     

A transposable element that splits the promoter region inactivates a Drosophila cuticle protein gene.

Two mutations that affect larval cuticle protein gene expression in the 2/3 variant Drosophila melanogaster strain were investigated. We demonstrate that this strain synthesizes an electrophoretic variant, fast 2 (CPf2), of wild-type cuticle protein 2(CP2). It also lacks detectable amounts of cuticle protein 3 (CP3). The other major cuticle proteins are still present. Protein and DNA sequence analyses indicate that point mutations cause two amino acid substitutions that change the electrophoretic mobility of CPf2 relative to that of CP2. The mutation abolishing the expression of CP3 was found to be a 7.3-kilobase DNA insertion located within the T-A-T-A box region of this gene, at -31 base pairs from the mRNA start site. This DNA insertion, called H.M.S. Beagle, belongs to a conserved family of repeated DNA elements that have characteristics similar to those of previously characterized Drosophila transposable elements. H.M.S. Beagle elements are repeated approximately 50 times in the haploid genome and exhibit restriction fragment-length polymorphisms around points of insertion between Canton S, Oregon R, and 2/3 Drosophila strains. Sequence analysis indicates that H.M.S. Beagle contains 266-base-pair direct repeats at its termini and is flanked by a duplication of 4 base pairs of target DNA sequence, T-A-T-A, in the CP3 gene insertion. Thus, insertion of a transposable element into the putative promoter region of the CP3 gene is evidently responsible for inactivating CP3 gene expression.     

Analysis of a transposable element in Caenorhabditis elegans.

A transposable element, designated Tc1, has been characterized in Caenorhabditis elegans. Tc1 is 1.7 kilobases long, has an inverted terminal repeat of less than 100 base pairs, and is repeated as a highly conserved element. The copy number and genomic positions of Tc1 are extremely variable among strains, implying that Tc1 is mobile. However, progeny of interstrain crosses did not show hybrid dysgenic traits that might be due to Tc1 transposition.