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Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations.

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.

Blastocyst↗

Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene.

The interaction in vitro of the RNA of the Q-strain of cucumber mosaic virus (CMV) with its satellite RNA (sat-RNA) has been studied. In hybridisation reactions containing 30% formamide at 45 degrees, sat-RNA binds to CMV RNA 3 and 4 but not to CMV RNA 1 and 2 or RNA from tobacco mosaic virus and alfalfa mosaic virus. The viral coat protein gene present in RNA 3 and 4 contains the site of binding but this region does not contain complementary sequences of any significant length to the sat-RNA sequence. However, the optimum alignment of short complementary sequences present in these regions revealed a stable structure in which it is proposed that sat-RNA twists around the coat protein gene so that two separate blocks of nucleotides in sat-RNA base pair in opposite directions with two adjacent blocks in the coat protein gene to form a knot-like structure. The binding site is a region of 33 nucleotides within the coding region of the coat protein gene which base pairs with residues 98-113 and 134-152 of sat-RNA. The possibility of the binding region of sat-RNA functioning as an "anti-sense" sequence in regulation of the viral coat protein synthesis is discussed.

Base Sequence↗

Diffuse plaques in the striatum in Alzheimer disease (AD): relationship to the striatal mosaic and selected neuropeptide markers.

Although neuritic and diffuse plaques generally coexist in Alzheimer disease (AD) neocortex, the predominance of diffuse plaques in the striatum prompted us to explore the potential influence of striatal features such as the striatal mosaic on beta-amyloid (A beta) deposition. Using double immunohistochemistry with an antibody to A beta in combination with antibodies to met-enkephalin or somatostatin, we investigated the relationship between diffuse plaques and neuropeptide distribution in the dorsal striatum. This relationship was examined in "pure" AD cases as well as in AD cases with coexisting Parkinson disease (PD) pathology, i.e. nigral degeneration and Lewy bodies at any site (AD + PD). Despite the presence of numerous A beta-positive diffuse plaques in both groups, the mosaic pattern, as exemplified by met-enkephalin-immunoreactive patches, was preserved. No obvious association was observed between the plaques and met-enkephalin-positive patches or somatostatin-immunoreactive neurons. Tyrosine hydroxylase immunoreactivity in the matrix was, however, diminished in AD + PD, most likely reflecting the nigral degeneration in these cases. Overall, these observations suggest that neostriatal A beta deposition in AD is not influenced by environmental factors associated with the striatal mosaic.

Aged↗

Tobacco mosaic virus particle structure and the initiation of disassembly.

The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface.

Computer Simulation↗

Coat protein gene sequences of two cucumber mosaic virus strains reveal a single amino acid change correlating with chlorosis induction.

The coat protein genes of two chlorosis-inducing strains of cucumber mosaic virus (CMV) were compared by nucleotide sequence analysis. The predicted amino acid sequences of the encoded coat proteins were compared with those of two other chlorosis-inducing and four mosaic-inducing CMV strains. Overall, the sequences were highly conserved, with more than 95% amino acid sequence identity between any two strains. However, a proline is present at amino acid 129 of all the mosaic-inducing strains, whereas that position is occupied by either a serine or a leucine in the coat proteins of all the chlorosis-inducing strains. The correlation of chlorosis induction and a substitution for proline with leucine or serine at amino acid 129 suggests that this residue is the determinant of chlorosis induction.

Amino Acid Sequence↗

Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic.

A combination of the polymerase chain reaction (PCR), asymmetric PCR (A-PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV-DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A-PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A-PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.

Base Sequence↗

The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain. CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.

Amino Acid Sequence↗

Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

Amino Acid Sequence↗

Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon.

Stem cuttings were collected in Cameroon from cassava plants displaying cassava mosaic disease (CMD) symptoms. The nature of the viruses present was determined by using the PCR with primers specific for the coat protein (CP) genes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). All samples were infected by ACMV and eight of the 50 samples were infected by both ACMV and an EACMV-like virus. The complete nucleotide sequences of DNA-A and -B of representative ACMV and EACMV-like viruses were determined. The DNA-A component of the EACMV-like virus contained evidence of recombination in the AC2-AC3 region and DNA-B also contained evidence of recombination in BC1. However, both components retained gene arrangements typical of bipartite begomoviruses. When Nicotiana benthamiana plants were doubly inoculated with these Cameroon isolates of ACMV and EACMV (ACMV/CM, EACMV/CM) by using sap from cassava plants or infectious clones, the symptoms were more severe than for plants inoculated with either virus alone. Southern blot analysis of viral DNAs from infected plants showed that there were significantly higher levels of accumulation of both ACMV/CM components and, to a lesser extent, of EACMV/CM components in mixed-infected plants than in singly infected plants. These results strongly suggest the occurrence of a synergistic interaction between the two viruses.

Base Sequence↗

Mutational evidence that the VPg is involved in the replication and not the movement of Pea enation mosaic virus-1.

Pea enation mosaic disease is caused by an obligatory association between the enamovirus Pea enation mosaic virus-1 (PEMV-1) and the umbravirus Pea enation mosaic virus-2 (PEMV-2). Encapsidated RNAs 1 and 2 are covalently linked to a 3138 Da VPg encoded by the RNA of PEMV-1. To determine the role of the VPg in the pathogenicity of PEMV (PEMV-1+PEMV-2), the infectivity of clones with mutations in key amino acids in the VPg was evaluated in protoplasts and in plants. Using quantitative, real-time RT-PCR, we concluded that the inability of certain mutants to infect plants was due to their replicative (and not their movement) incompetence. Mutant clones that produced delayed and less severe infections accumulated 10- to 100-fold less RNA-1 compared to WT-RNA-1 both in plants and in protoplasts. The RNAs of clones that produced WT-like infections accumulated to levels similar to those of WT-PEMV. Also, we demonstrate that the severity of symptoms produced by WT-PEMV is proportional to the amount of RNA-1 that accumulates in infected plants and seems to be independent of the amount of RNA-2. A dual role for the VPg in the pathogenicity of PEMV is proposed.

Amino Acid Sequence↗

Diversity of mosaic structures and common ancestry of human immunodeficiency virus type 1 BF intersubtype recombinant viruses from Argentina revealed by analysis of near full-length genome sequences.

The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.

Argentina↗

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement.

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.

Cucumovirus↗

Clonal origin of gamma-glutamyl transpeptidase-positive hepatic lesions induced by initiation-promotion in ornithine carbamoyltransferase mosaic mice.

Since the deficiency of ornithine carbamoyltransferase (OCT) is inherited as an X-linked dominant trait in sparse-fur with abnormal skin and hair (Spf-ash) mice, the livers of heterozygous Spf-ash females show mosaicism in regard to OCT. We induced enzyme-altered foci and nodules, presumptive preneoplastic lesions for hepatocellular carcinomas, in the livers of OCT mosaic mice (Spf-ash x C3H F1), and investigated the clonality of the lesions. Simultaneous histochemical staining for OCT and gamma-glutamyl transpeptidase (GGT) demonstrated that all GGT-positive lesions (ranging in size from 3 cells to a few millimeters in diameter) were either positive or negative for OCT, and no mosaic lesions were detectable. The results indicate that individual enzyme-altered hepatocytic lesions are the result of clonal proliferation.

Animals↗

First description of somatic mosaicism in MYH9 disorders.

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Dohle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, which encodes non-muscle myosin heavy chain-A (NMMHCA). These disorders are known to be transmitted in an autosomal dominant manner, although about 20% of cases are considered to be sporadic. We report here the first case of a MYH9 disorder because of somatic mosaicism. The patient was the father of a male with typical May-Hegglin anomaly. The father had normal platelet counts, however, both normal-sized and giant platelets were observed on his peripheral blood smears. In addition, 14% of neutrophils contained inclusion bodies and the rest showed a normal morphology. Quantitative fluorescent polymerase chain reaction analysis showed that only 6% of DNA from peripheral blood leucocytes harboured the mutation. The mosaicism was demonstrated at a similar rate in different tissues, buccal mucosa cells and hair bulb cells, implying that the mutation had occurred before gastrulation. Mosaicism might account for some de novo mutations in MYH9 disorders.

Base Sequence↗

Incomplete prenatal diagnosis of G-trisomy mosaicism.

Chromosomal mosaicism is difficult to detect in prenatal diagnosis, and it seems even more difficult to predict its significance correctly. We present a case, in which G-trisomy mosaicism was diagnosed prenatally but was not verified in the aborted fetus. A practical approach to suspected fetal mosaicism in prenatal diagnosis is proposed.

Adult↗

Trisomy 18 mosaicism: clues to the diagnosis.

Karyotypes of blood and skin fibroblasts at ages 3 and 8.5 years had shown non-mosaic trisomy 18 in a male now of age 19. Because of his prolonged survival and an atypical phenotype, skin fibroblast cultures from a new biopsy were established at age 18, and only normal 46,XY cells were observed, while peripheral blood lymphocytes still demonstrated 47,XY, + 18. This patient and six others with trisomy 18 mosaicism illustrate the advisability of looking for such a pattern in individuals whose phenotype in early life is not fully consistent with the trisomy 18 syndrome. Additional clues to the presence of trisomy 18 mosaicism are male sex, survival beyond 2 years and lack of fingertip arches.

Adult↗

Trisomy 10 mosaicism in a newborn boy; delineation of the syndrome.

The fifth case of trisomy 10 mosaicism is presented. Only in cultured fibroblasts this mosaicism was found, while peripheral lymphocytes revealed a normal karyotype. In comparison with the literature, trisomy 10 mosaicism syndrome is further delineated compromising of failure to thrive, high forehead, hypertelorism, mongoloid eye slant, blepharophimosis, dysplastic, large ears, retrognathia, long slender trunk, marked plantar and palmar furrows, cardiopathy and early death.

Abnormalities, Multiple↗