DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

Tomographic distribution of acetylated histone H4 in plant chromosomes,nuclei and nucleoli

Root tip cells of broad bean (Vicia faba L. cv. ’Wase soramame’) and barley (Hordeum vulgare L. cv. ’Minorimugi’) were immunostained with antibodies specific for acetylated histone H4. With an antiserum that recognizes histone H4 acetylated at lysine-5, the nucleolar organizing region (NOR) in mitotic chromosomes was strongly labeled in both species. The broad bean had two signals in the metaphase and telophase chromosome complements and four signals in the prophase and anaphase chromosome complements, while the barley had four signals in the metaphase and telophase chromosome complements and eight signals in the prophase and anaphase complements. Five different patterns of signals were observed at interphase: in type I only nucleoli were wholly stained; in type II perinucleolar knob-like signals and/or fiber-like signals emanated from the nucleus; in type III aggregate signals appeared in the nucleolus; in type IV many small dot-like signals were distributed throughout the nucleus, except nucleoli; and in type V string-like or some granule-like signals appeared in the nucleoli. Type II was very similar to previous results by in situ hybridization with sense rDNA probes. Type III was similar to the patterns of DNA synthesis recognized as chromatin domains by anti-BrdU antibodies. Type V was very similar to the results of in situ hybridization with pTa71, rDNA probes and the appearance of the dense fibrillar components of the nucleolus. Received: 7 August 1996; in revised form: 16 September 1996 / Accepted: 16 September 1996     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Somatic mosaicism for deletion of the entire NF1 gene identified by FISH

We report a young child with a large congenital cervical plexiform neurofibroma and multiple café-au-lait spots in a generalized distribution who has mosaicism for complete deletion of the NF1 gene. The deletion was demonstrated with intragenic cosmid probes as well as YACs spanning a 700-kb contig including NF1, by two-color FISH with an NF1 and a control probe. Using different intragenic probes, deletion was found in 77–84% of cultured peripheral blood lymphocytes but not in cultured skin fibroblasts. Neither parent has signs of neurofibromatosis type 1 (NF1) or a gene deletion. This is the first report of mosaicism for complete deletion of the NF1 gene. The child did not have typical NF1 or display segmental features of NF1. Received: 6 June 1996 / Revised: 2 October 1996     

Identifying different types of de-differentiated microspores from indica-japonica F1 hybrids with subspecies-differentiating RFLP probes in rice

The indica, japonica and intermediary types of de-differentiated microspores from indica-japonica hybrids were identified with 11 subspecies-differentiating RELP probes in rice (Oryza sativa L.). The results showed that the distribution of indica, japonica and intermediary types of de-differentiated microspores could be easily detected in a simple and quick way using the RFLP method. Moreover, the microspores from the same hybrid but inoculated onto different media, or microspores from different hybrids when inoculated onto the same medium, often displayed distinctive distribution curves of de-differentiated microspores types, indicating that the media employed in this experiment had high selectivity for the de-differentiation of certain types of microspores. The application of the RELP method to de-differentiated microspore identification is of great theoretical and practical significance in rice doubled-haploid breeding. Received: 27 February 1996 / Accepted: 14 June 1996     

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen.This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS_AH) or of CSA (CSA_AH), and 2) binding of CPS_AH to CSA or of CPS to CSA_AH. Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA_AH (EDC:CPS-CSA_AH) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum.Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.     

Chromosomal evolution of the Chinese muntjac (Muntiacus reevesi)

The aim of this study was to test the validity of the hypothesis that the 2n=46 karyotype of the Chinese muntjac (Muntiacus reevesi) could have evolved through 12 tandem fusions from a 2n=70 hypothetical ancestral karyotype, which is still retained in Chinese water deer (Hydropotes inermis) and brown-brocket deer (Mazama gouazoubira). Combining fluorescence-activated chromosomal sorting and degenerate oligonucleotide-primed polymerase chain reaction, we generated chromosome-specific DNA paint probes for 13 M. gouazoubira chromosomes and most of the M. reevesi chromosomes with the exception of 18, 19 and X. These paint probes were used for fluorescence in situ hybridisation to chromosomal preparations of M. reevesi, H. inermis and M. gouazoubira. Chromosome-specific paint probes from M. reevesi chromosomes 1–5 and 11 each delineated more than one homologous pair (18 pairs in total) on the metaphases of H. inermis and M. gouazoubira. All the other probes from M. reevesi and probes from M. gouazoubira each hybridised to one pair of homologous chromosomes or regions. The C5 probe, derived from centromeric satellite sequences of M. reevesi, hybridised to the centromeric regions of all chromosomes of these three species. Most interestingly, several non-random interstitial signals, which are apparently localised to the putative fusion points, were found on chromosomes 1–5 and 11 of M. reevesi. Both the reciprocal painting patterns and localisation of the C5 probe demonstrate that M. reevesi chromosomes 1–5 and 11 could have evolved from 18 different ancestral chromosomes through 12 tandem fusions, thus providing direct molecular cytogenetic support for the tandem fusion hypothesis of karyotype evolution in M. reevesi. Received: 10 October 1996; in revised form: 18 December 1996 / Accepted: 27 December 1996     

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

UV irradiation as a tool for obtaining asymmetric somatic hybrids between Nicotiana plumbaginifolia and Lycopersicon esculentum

UV-irradiated kanamycin-resistant Lycopersicon esculentum leaf protoplasts were fused with wild-type Nicotiana plumbaginifolia leaf protoplasts. Hybrid calli were recovered after selection in kanamycin-containing medium and subsequently regenerated. Cytological analysis of these regenerants showed that several (2–4) tomato chromosomes, or chromosome fragments, were present in addition to a polyploid Nicotiana genome complement. All lines tested had neomycin phosphotransferase (NPTII) activity and the presence of the kanamycin gene was shown by Southern blotting. In two cases a different hybridization profile for the kanamycin gene, compared to the tomato donor partner, was observed, suggesting the occurence of intergenomic recombination events. The hybrid nature of the regenerants was further confirmed by Southern-blotting experiments using either a ribosomal DNA sequence or a tomato-specific repeat as probes. The hybrids were partially fertile and some progeny could be obtained. Our results demonstrate that UV irradiation is a valuable alternative for asymmetric cell-hybridization experiments. Received: 3 August 1996 / Accepted: 23 August 1996     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

Beyond the Wobble: The Rule of Conjugates

A reexamination of the genetic code suggests a rule of conjugates which captures the observed quartet degeneracies without exception. Adenine is the conjugate of cytosine and uracil is the conjugate of guanine. Further analysis reveals that the rule of conjugates is a macrolevel manifestation of the molecular-level hydrogen-bonding and base-stacking interactions at the decoding site. This new perspective is of significance to evolutionary discussions of nucleic acid bases, genetic code, and interactions involving RNAs. Received: 29 November 1996 / Accepted: 8 June 1997     

Hydrophobized triphenyl phosphonium derivatives for the preparation of mitochondriotropic liposomes: choice of hydrophobic anchor influences cytotoxicity but not mitochondriotropic effect

Context: Nanocarrier-based strategies to achieve delivery of bioactives specifically to the mitochondria are being increasingly explored due to the importance of mitochondria in critical cellular processes.

Objective: To test the ability of liposomes modified with newly synthesized triphenylphosphonium (TPP)–phospholipid conjugates and to test their use in overcoming the cytotoxicity of stearyl triphenylphosphonium (STPP)-modified liposomes when used for delivery of therapeutic molecules to the mitochondria.

Methods: TPP–phospholipid conjugates with the dioleoyl, dimyristoyl or dipalmitoyl lipid moieties were synthesized and liposomes were prepared with these conjugates in a 1?mol% ratio. The subcellular distribution of the liposomes was tested by confocal microscopy. Furthermore, the liposomes were tested for their effect on cell viability using a MTS assay, on cell membrane integrity using a lactate dehydrogenase assay and on mitochondrial membrane integrity using a modified JC-1 assay.

Results: The liposomes modified with the new TPP–phospholipid conjugates exhibited similar mitochondriotropism as STPP-liposomes but they were more biocompatible as compared to the STPP liposomes. While the STPP-liposomes had a destabilizing effect on cell and mitochondrial membranes, the liposomes modified with the TPP–phospholipid conjugates did not demonstrate any such effect on biomembranes.

Conclusions: Using phospholipid anchors in the synthesis of TPP–lipid conjugates can provide liposomes that exhibit the same mitochondrial targeting ability as STPP but with much higher biocompatibility.     

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I)

In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1–2, and 2. Received: 20 March 1996 / Revised: 14 May 1996     

A 25-kb deletion in the 5′ region of the cytochrome b558 heavy chain gene (CYBB) in a patient with X-linked chronic granulomatous disease

We performed molecular genetic analyses of the family of a boy suffering from chronic granulomatous disease (CGD) after immunocytochemically confirming him and his mother to be an X-linked CGD patient and a mosaic carrier, respectively. Southern blot hybridization using cDNA for the cytochrome b₅₅₈ heavy chain gene (CYBB) as a probe showed that the patient had a deletion in the 5′ region of the CYBB and his phenotypically normal mother was heterozygous for this deletion. Polymerase chain reaction analyses of all 13 exons of the patient’s CYBB gene demonstrated that the deletion extends from exon 7 or neighboring introns to 5′ upstream. The length of the deletion was determined by pulsed-field gel electrophoresis and Southern blotting of genomic DNA using CYBB cDNA and the genetic marker pERT55-5, centromeric to CYBB, as probes. Both probes recognized common SfiI-NotI fragments of 120 kb and 95 kb in normal individuals and the patient, respectively. These results revealed that the patient has a 25-kb deletion spanning from the middle of CYBB to 5′ upstream. This is the only report of a large 5′ deletion in CYBB and also the first observation that CYBB and pERT55-5 are within 120 kb in Xp21. Received: 24 September 1996 / Revised: 10 November 1996     

Asymmetric fusion between wild and cultivated species of potato (Solanum spp.) –detection of asymmetric hybrids and genome elimination

 The objective of this study was to evaluate the suitability of different techniques for a simple and rapid identification of asymmetric hybrids, without the use of selection markers and independent of the fusion partners used. Additionally, the degree of donor DNA elimination was determined. Among 473 viable plants obtained from asymmetric fusion experiments between three di-haploid breeding lines of potato (Solanum tuberosum) and diploid wild species (S. bulbocastanum, S. circaeifolium; X-ray treatment of the wild species) the most promising ones were investigated with three different methods: flow cytometry, RFLP analysis with an oligonucleotide probe (GATA)₄, and with single-copy probes. Flow cytometry, which combines a high screening capacity with detailed information about the DNA content and allows a distinction between asymmetric hybrids and chimeras, detected 31 hypo-tetraploid and 42 hypo-hexaploid regenerates among 224 plants. With the oligonucleotide probe (GATA)₄ only a few asymmetric hybrids were detected among all regenerates. More than 50% of these asymmetric regenerates were chimeras. Concerning the degree of DNA elimination, the results obtained by RFLP analysis with 17 single-copy probes were correlated with the results obtained by flow cytometry. The maximum DNA elimination of the donor genome was 52%. As a trend, an irradiation dosage of 210 Gy caused a higher DNA elimination in the wild species than a dosage of 70 Gy. No calli were obtained after irradiation of the wild species with 420 Gy. Received : 6 December 1996/Accepted: 24 January 1997     

Comparison of UV light, X-ray and restriction enzyme treatment as tools in production of asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana

 Asymmetric somatic hybrids between Brassica napus (receptor) and Arabidopsis thaliana (donor) have been produced by three different methods supposed to induce asymmetry. The donor protoplasts were either UV- or X-irradiated, or the mixture of protoplasts was treated with the restriction enzyme PvuII immediately before fusion. The genome composition of the hybrids was analysed with Southern blot hybridisations using 15 different mapped A. thaliana RFLP markers as probes. Both UV- and X-irradiation were found to be efficient treatments for induction of asymmetry in somatic hybrids in a dose-dependent manner. The addition of a restriction enzyme to the protoplast mixture did not have any effect on the frequency of asymmetric hybrids or on the degree of asymmetry in the hybrids produced. UV- and X-irradiation resulted in higher fertility in the hybrids, while PvuII treatment did not have any effect on seed set. A significant positive correlation between degree of asymmetry in different plants and seed set after selfing was detected. Received: 18 December 1997 / Accepted: 7 January 1998     

Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis.