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Complex chromosomal rearrangements: some breakpoints may have cellular adaptive significance.

Cytogenetic study of a 3-year-old girl with developmental delay and some minor abnormalities revealed a complex chromosome rearrangement (CCR) involving seven chromosomes with eight breakpoints, leading to monosomy of segment 5q15-q22. According to breakpoint distribution, CCRs may be classified as those with primary intrachromosomal abnormalities (including inversions, insertions, duplications, etc.) and those without them. Only the latter group of CCRs was used in this analysis. Comparison of theoretical and observed breakpoint distributions in 33 cases demonstrated that recurrent involvement of some chromosome(s) ("re-entry") occurs more frequently than expected. One possible explanation for this observation suggests that the initial event leads to an unstable provisional rearrangement, and subsequent breaks are necessary to stabilize the karyotype.

Child, Preschool↗

Construction of phospholamban antisense RNA recombinant adeno-associated virus vector and its effects in rat cardiomyocytes.

AIM: To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), and analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and the change of intracellular free Ca2+ concentration ([Ca2+]i) in rat cardiomyocytes. METHODS: The target gene encoding PLB antisense RNA was inserted inversely into the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The recombinant plasmid and pAAV-RC and pHelper were co-transfected into 293 cell. At the same time, a viral production positive control (rAAV-LacZ) and negative control were performed. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site beta-Galactosidase staining were performed to observe the transfer efficiency. Reverse transcription-PCR and Western blot were used to determine the mRNA and protein expression of PLB and SERCA. The activity of SERCA and the [Ca2+]i were measured. RESULTS: The rAAV vectors were constructed successfully and were transfected into rat cardiomyocytes effectively. The PLB mRNA and protein expression were reduced in rat cardiomyocytes transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca2+]i in the rAAV-asPLB transfected group decreased. The level of [Ca2+]i increased when induced by isoproterenol. CONCLUSION: AAV-asPLB vector was constructed successfully, which disrupted the expression of PLB, enhanced the activity of SERCA, reduced the resting [Ca2+]i, and improved the cardiac function.

Animals↗

Divergent orientation of transcription from the arginine gene ECBH cluster of Escherichia coli.

Ribonucleic acid (RNA) isolated from Escherichia coli W3350 (F(-), argE(+)C(+)B(+)H(+)), in the absence of l-arginine, hybridizes with the separated leftward (l) and rightward (r) transcribing strands of the arginine transducing phage hphi80dargE(+)C(+)B(+)H(+)ppc(+)imm(lambdacI857) deoxyribonucleic acid (DNA) with a ratio of 30:70, respectively. In the presence of l-arginine and its intermediates, l-ornithine and l-citrulline, RNA transcriptions from both the strands of the argECBH cluster were repressed. The derepressed RNA, when hybridized with the separated strands of hphi80dargEC-I imm(lambda) phage DNA (the arginine genes are inversely inserted in this phage), which has a deletion in gene E and extends to gene C of the argECBH cluster, showed no leftward transcription, whereas the rightward transcription was reduced to about 40% of that when the DNA carrying the entire ECBH cluster was used for hybridization. The hybridization results thus demonstrate that (i) the regulation of the argECBH gene cluster in E. coli is under transcriptional control, (ii) the orientation of transcription is divergent, (iii) E gene transcribes anticlockwise, whereas the rest of the genes, C, B, and H, transcribe clockwise, and (iv) the position of the promoter(s) and operator(s) is located between the E and C genes of the argECBH cluster.

Arginine↗

Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.

Blotting, Southern↗

Molecular breeding of allergy vaccines and antiallergic cytokines.

Molecular breeding, also called DNA shuffling, is a technology that enables the generation of large libraries of novel genes and vectors, from which improved variants can be selected based on functional properties. In a common format, it involves recursive recombination and mutation, performed by random fragmentation of related DNA sequences, followed by reassembly of the fragments in a self-priming polymerase chain reaction. As in natural evolution, the technique takes advantage of crossovers, deletions, insertions, inversions and point mutations of genes to generate large pools of related sequences. Molecular breeding can be used to generate improved variants of proteins used as therapeutics, such as vaccine antigens, growth factors and immunomodulatory molecules. Moreover, the technology can be applied to evolve entire viruses or vectors, including DNA vaccines. Cytokines downregulating allergic immune responses and allergens are attractive targets for evolution by molecular breeding. This review describes approaches to generate chimeric allergens with T cell epitopes from multiple allergen homologues, while reducing the recognition by preexisting IgE. In addition, the results and applications of molecular breeding in the evolution of improved antiallergic cytokines are discussed.

Allergens↗

Report of the committee on structural chromosome changes in neoplasia.

An enormous amount of data on neoplasia-associated chromosome aberrations has accumulated over the past two years. More than 4,000 tumors with a chromosome anomaly identified by banding have been published since HGM9, and the total number of cases contained in the registry on which the Catalog of Chromosome Aberrations in Cancer (Mitelman, 1988) is based is now well above 12,000. The information presently available is, however, still in many respects incomplete. First, the data is heavily biased in favor of hematologic disorders. Solid tumors comprise only 20% of the total data base, which is totally disproportionate to the relative contribution of these disorders to human cancer morbidity and mortality. For example, malignant epithelial tumors (carcinomas), which cause almost 80% of all cancer deaths in man, comprise only 7% of the total. Second, our knowledge about early stage tumors is very limited. For example, the great majority of the solid tumors that have been studied cytogenetically have been metastatic lesions or effusions (advanced tumors usually have a large number of complex structural and numerical chromosome aberrations). Obviously, many more such neoplasms will have to be studied before the primary (pathogenetically essential) changes can be distinguished from the confusing variety of secondary abnormalities that may dominate the karyotype. It should be noted that secondary changes may also be nonrandom, and may be important for tumor progression. Therefore, no attempt has been made in this report to distinguish between primary and secondary changes. All nonrandomly occurring abnormalities that met the criteria for inclusion are listed in Table 1 irrespective of their presumed pathogenetic significance. Results of molecular genetic studies (e.g. the demonstration of loss of heterozygosity or gene amplification) were not considered, although they may be included in the HGM10.5 report. A total of 149 nonrandom chromosome changes were identified (Table 1) in 43 different types of neoplastic disorders, including hematologic diseases and malignant lymphomas, as well as tumors of epithelial, mesenchymal, neurogenic, germ cell, and melanocytic origin. The aberrations comprise a variety of structural chromosome rearrangements (translocations, inversions, insertions, deletions, duplications and isochromosomes), and all chromosomes, except the Y chromosome are involved. The great majority (121 of the 149 identified aberrations) represent well-defined, specific structural changes. More than half of them are consistently associated with a particular morphologic disease characteristic. Twenty-eight of the aberrations, although nonrandom, are not characterized as well. Most are deletions or translocations that only affect a certain chromosome region, often spanning several bands.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromosome Aberrations↗

Strategies for the detection of copy number and other structural variants in the human genome.

Advances in genome scanning technologies are revealing that copy number variants (CNVs) and polymorphisms, ranging from a few kilobases to several megabases in size, are present in genomes at frequencies much greater than previously known. Discoveries of additional forms of genomic variation, including inversions, insertions, deletions and complex rearrangements, are also occurring at an increased rate. Along with CNVs, these sequence alterations are collectively known as structural variants, and their discovery has had an immediate impact on the interpretation of basic research and clinical diagnostic data. This paper discusses different methods, experimental strategies and technologies that are currently available to study copy number variation and other structural variants in the human genome.

Gene Dosage↗

Homozygous chromosomal aberrations in Anopheles stephensi.

Homozygotes for six autosomal paracentric inversions, an inserted paracentric inversion, an autosomal translocation, and two X-chromosome-chromosome 3 translocations in Anopheles stephensi are described. Three of these aberrations are being maintained in pure strains without the necessity of selection.

Animals↗

Karyotypic analysis of seven established human esophageal carcinoma cell lines.

Fifty metaphases from each of seven esophageal carcinoma cell lines (TE-1, TE-4, TE-5, TE-6, TE-9, HCE-6, and HCE-8) were karyotypically analyzed for both numerical and structural chromosomal abnormalities. The modes of numerical variation found in the TE cell lines were as follows: 1) a wide range of variation in chromosome numbers (45-100)/cell with a modal number of 52 (TE-1); and 2) a narrow range of variation at the hypertetraploidy level (TE-4), hypotriploidy level (TE-5 and TE-6) and hypertriploidy level (TE-9). The distribution of chromosomal number/cell in cell lines HCE-6 and HCE-8 was extremely diverse. It was difficult to find a modal number in the range of 94-117 in the HCE-6 line or in the range of 45-52 in the HCE-8 line. The structural changes were extensive in all of these cell lines. Chromosomes most frequently involved in the structural changes were 1, 2, 3, 6, 7 and 9. Most of these abnormalities resulted from simple deletion, duplication, insertion, inversion or translocation. A few of them were complicated rearrangements. No breakpoint was shared by all of the cell lines.

Chromosome Aberrations↗

[The karyotypic variability of Chinese hamster CHLV-79 RJK cells characterized by multiple drug resistance resulting from the amplification of the mdr gene family].

Variability in karyotype structure of Chinese hamster lung V-79 RJK cells and of their six cell sublines, selected for increasing concentrations of ethidium bromide (EB), was investigated in addition to the number of mdr gene copies in cells, both EB sensitive and resistant. It is shown that EB resistant cells exhibit cross-resistance to different drugs resulting from mdr genes amplification. Southern DNA blot hybridization has shown that in Vebr-2 cells (the 1st step of selection) the number of mdr gene copies increased by 10 times, whereas in Vebr-30 cells (the 6th step of selection) the number of mdr gene copies remained the same as in Vebr-2 cells. The level of mdr genes expression in Vebr-30 cells being higher than in Vebr-2 cells. In Vebr-2 cells, homogeneously and differentially stained regions (HSRs) were detected in loci 1p31 and 1q26 of chromosome 1 material (markers Z1 and Z6, respectively). On the following selection steps (prolonged cultivation or increased drug concentration) additional HSRs appeared in chromosome 2 (locus 2qter), in derivatives of chromosome 5 (marker Z7, locus Z7pter) and chromosome X (marker Z2, locus Z2qter). In the course of prolonged cultivation, chromosome 2 and derivatives of chromosomes 1, 2, 5 and X, in which HSRs were found, participated in the formation of new markers resulting from deletions, inversions, insertions and translocations of the chromosomal material. It is supposed that mdr genes amplification in V-79 RJK cells resistant to EB may be regarded as a factor inducing subsequent genome destabilization and eventual progressive changes in the karyotype structure.

Animals↗

Intrachromosomal insertion mimicking a pericentric inversion: molecular cytogenetic characterization of a three break rearrangement of chromosome 20.

Intrachromosomal insertions are uncommon rearrangements, in which a chromosomal segment is intercalated into another part of the same chromosome. The insertion may occur in the same arm (paracentric) or in the other arm (pericentric). The cytogenetic recognition of these structurally rearranged chromosomes can be difficult, and intrachromosomal insertions can be easily mistaken for inversions. We describe a case of a familial pericentric insertion of chromosome 20, initially misdiagnosed as a pericentric inversion in the healthy carrier and then reinterpreted as insertion in an abnormal child with a recombinant chromosome. Fluorescence in situ hybridization (FISH) allowed us to confirm the mechanism of recombinant formation and to locate the three breakpoints precisely. Our cytogenetically unbalanced epileptic patient carried a 20q deletion and 20p duplication, and the genes, CHRNA4 and KCNQ2 that have been implicated in autosomal dominant epilepsy, were deleted. The haplo-insufficiency of these two genes may contribute to the cause of epilepsy in patients with ring chromosome 20.

Abnormalities, Multiple↗

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

Arabidopsis↗

Unusual mutations in Btk: an insertion, a duplication, an inversion, and four large deletions.

Mutations in Bruton's tyrosine kinase (Btk) result in the immunodeficiency X-linked agammaglobulinemia (XLA). In a previous study of 101 patients with presumed XLA, we identified seven patients with large genomic alterations in Btk. The recent completion of 100 kb of contiguous DNA sequence at the Btk locus has allowed us to characterize these mutations in detail and to identify four different types of alterations. These alterations included a 253-bp retroposon insertion at position +5 within intron 9, an inversion of greater than 48 kb that disrupted Btk between exons 4 and 5, a 12.9-kb duplication including Btk exons 2 to 5, and four deletions ranging from 2.8 to 38 kb in size. The duplication and three of the deletions resulted from unequal crossovers of Alu repeats. Further, three of the deletions terminated within a repeat-rich cluster spanning 30 kb of sequence 3' of Btk exon 19, suggesting that this region was more susceptible to unequal crossovers than the rest of the Btk gene. These studies describe the first reports of an insertion, an inversion, and a duplication in Btk and demonstrate the utility of large-scale sequencing in the elucidation of disease-causing mutations.

Agammaglobulinaemia Tyrosine Kinase↗

Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks.

Rejoining of DNA double-strand breaks (DSB) plays a central role in the various processes leading to DNA rearrangements. We have analyzed DNA alterations induced by restriction enzymes that produce DSB with specific types of ends. Restriction enzymes were electroporated into a human lymphoblastoid cell line that stably maintains pHAZE, an EBV-based vector containing the lacZ gene. After allowing time for DSB repair, pHAZE DNA was rescued and screened in Escherichia coli. Mapping and sequence analysis of mutant copies of pHAZE indicated that restriction enzymes induced all classes of alterations except base substitutions (base deletions and insertions, large-scale deletions, inversions, and insertions). The spectra of alterations were distinctive for each enzyme and appear to be the consequence of specific end-modification processes.

Base Sequence↗

Hybrid dysgenesis-induced revertants of insertions at the 5' end of the rudimentary gene in Drosophila melanogaster: transposon-induced control mutations.

Mutations in the 5' control region of the rudimentary (r) gene of Drosophila melanogaster were generated by using hybrid dysgenesis to mobilize P elements that were already inserted within this region. Eighteen new mutations out of 7793 chromosomes were isolated. Among the mutations were small insertions, deletions and inversions. All five of the deletions deleted into the 5' coding region of the r gene resulting in a severe mutant phenotype. The inversions and small insertions left the coding region intact, but altered the 5' control region. Northern analyses of the message levels in adult females indicated that these mutations alter the amount of the wild-type rudimentary message. These data also indicated that there is a region about 800 bp upstream of the start of transcription which is necessary for normal r expression. This region is not involved with the temporal and spatial regulation of r gene expression, but with the quantitative expression. An analysis of the DNA changes associated with each of the mutations demonstrates that P elements usually mobilize in an imprecise manner. Of the 22 mutations that have been isolated to date, only three are precise excisions. This means that P elements are potent mutators of the genome.

Animals↗

Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations.

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.

Coliphages↗