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PubMed1990

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis.

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

Base Sequence↗

PubMed1988

The rosy region of Drosophila melanogaster and Drosophila simulans. I. Contrasting levels of naturally occurring DNA restriction map variation and divergence.

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.

Animals↗

PubMed1992

Mono- and bi-phasic Salmonella typhi: genetic homogeneity and distinguishing characteristics.

Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5' end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.

Africa↗

PubMed2026

Evolutionary dynamics of the chloroplast genome in Abutilon (Malvoideae, Malvaceae).

The genus Abutilon Mill. (Malvaceae) comprises approximately 178 species distributed across tropical and subtropical regions, many of which hold significant ornamental, economic, and medicinal value; yet its taxonomic classification remains challenging. In this study, six species were sequenced from herbarium specimens, and the chloroplast (cp.) genomes of ten additional species were assembled de novo from publicly available raw data. Three previously reported cp. genomes were also incorporated to characterise cp. genome structure, identify polymorphic loci, and perform phylogenetic analyses. The cp. genomes ranged from 159,458 to 160,454 bp and exhibited the typical quadripartite structure, with each genome containing 112 unique genes (78 protein-coding, 30 tRNA, and 4 rRNA) that showed conserved content and organisation. These genomes exhibited high similarity in GC content, inverted repeat boundaries, relative synonymous codon usage, amino acid frequencies, and substitution patterns. However, notable variation was observed in the total number of simple sequence repeats, ranging from 70 to 97 per genome. Selection analyses indicated predominant purifying selection, with evidence of episodic positive selection detected in rpoC2, rbcL, and ycf1. Two codons in rbcL were clade-specific and provided phylogenetic signal distinguishing Australian and Old World pantropical species. Nucleotide diversity analysis identified six highly polymorphic intergenic spacers (trnH-psbA, rps19-rpl2, psbT-pbf1, psaC-ndhD, trnR-atpA, and ndhJ-ndhK) that may be suitable for taxonomic studies. The phylogeny from maximum likelihood (ML) and Bayesian inference (BI) resolved two major clades: one comprising an exclusively Australian lineage occurring predominantly in arid and semi-arid environments, and the other a pantropical lineage spanning multiple continents. Abutilon grandifolium was recovered as sister to the remaining sampled Abutilon taxa in both ML and BI analyses, although no biogeographic origin inference can be drawn from this placement pending broader taxon sampling and integration of nuclear genomic data. These findings provide insights into the evolutionary dynamics of the cp. genome in Abutilon and offer a foundational genomic framework for refining Abutilon taxonomy.

Genome, Chloroplast↗

PubMed2026

Same Sex Chromosomes With Independent Origins in Haplochromine Cichlids.

Elucidating theories of sex chromosome evolution requires approaches that allow fine scale delimitations of sex-determining regions within a phylogenetic context. This can address whether shared sex chromosomes across related species are due to shared ancestry, or whether genetic sex-determining regions have repeatedly evolved. Haplochromine cichlids, as one of the most successful fish lineages on Earth, have been a focal study system of sex chromosome research, both because of their rapid rate of sex chromosome turnover and the repeated emergence of certain sex chromosomes across the lineage. Here, we newly describe sex chromosomes in members of the earliest branch of the modern haplochromines, the Tropheini, based on whole-genome sequencing data, using a combination of SNP- and kmers-based methods. We show that despite the repeated co-options of ancestral chromosomes LG5 and LG7 in these species, the origins of these sex chromosomes are independent. Investigation of gene functions, allele differences, and sex-biased gene expression within the discovered sex-linked regions provides no evidence that sexual antagonism has driven the repeated evolution of a region on LG5 that overlaps between four of these species. By comparing the sex-determining regions on LG5 and LG7 across haplochromines, we show that a common origin is unlikely, and that while sex chromosomes themselves may be shared between several Haplochromini, the sex-determining genes or mechanism likely differ. This study paves the way to explore newly emerging theories of sex chromosome evolution, such as the role of chromosomal fusion or recombination patterns across the genome.

Animals↗

PubMed1990

A simple and rapid method for HLA-DP genotyping by digestion of PCR-amplified DNA with allele-specific restriction endonucleases.

We previously reported a simple and rapid method for HLA-DQA genotyping by digestion of polymerase chain reaction-amplified DQA genes with allele-specific restriction endonucleases. Here we report the application of this method to DP genotyping. The second exon of the HLA-DPB genes was selectively amplified from genomic DNAs of 72 HLA-D homozygous B-cell lines by the polymerase chain reaction method. Amplified DNAs were digested with ApaI, SacI, BstUI, FokI, and RsaI, which can recognize allelic sequence variations in the polymorphic segments of the DPB second exon and then subjected to electrophoresis in polyacrylamide gels. Sixteen different polymorphic patterns of the restriction fragments were found, and twelve were identical to patterns predicted from the known DNA sequences correlating with each HLA-DPw specificity defined by cellular typing. The other four patterns were distinct from those of the known DPw specificities, suggesting the presence of novel DP alleles. This polymerase chain reaction-restriction fragment length polymorphism method provides a simple and rapid technique for accurate definition of HLA-DP types at the nucleotide level, replacing the technically demanding method of primed lymphocyte typing.

Alleles↗

PubMed2004

Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse.

Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7.

Adenylyl Cyclases↗

PubMed1985

Identification of a partial cDNA clone for the human receptor for complement fragments C3b/C4b.

Redundant oligonucleotides were synthesized based on amino acid sequences of tryptic peptides from the purified receptor for human complement fragments C3b/C4b (CR1). These probes were used to screen a size-selected human tonsilar cDNA library. A single positive clone was identified that hybridized to three oligonucleotide probes. The cDNA insert was 1.5 kilobases in length and contained sequences homologous to those of the oligonucleotide probes as well as nucleotide sequences corresponding to another independent CR1 tryptic peptide. Blot-hybridization analysis using fragments of the cDNA insert as probes revealed two distinct species of the CR1 message of 9 and 11 kilobases in human tonsil mRNA. The two EcoRI fragments of the CR1 cDNA insert hybridized to each other, suggesting the presence of homologous sequences. When used as probes in Southern blot analysis of human DNA, each fragment identified similar but not identical patterns of multiple restriction fragments, indicating either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes. Furthermore, an additional BamHI fragment was found to segregate with the expression of the S allotype of the CR1 protein in a family. Thus, the molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences.

Base Sequence↗

PubMed1987

Polymorphism and gene conversion in mouse alpha-globin haplotypes.

We have cloned and characterized three distinct alpha-globin haplotypes obtained from inbred strains of the mouse, Mus domesticus. We report here the complete nucleotide sequence of the six alpha-globin genes that the haplotypes contain. Our analysis of these genes and those from one other previously described haplotype indicates that recurrent gene conversion events have played a major role in their history. The pattern of nucleotide substitutions suggests that conversions have occurred both within and between haplotypes. Limited segments of coding and noncoding DNA have been involved in these gene conversion events. In two of the haplotypes, the nonallelic genes of each maintain DNA sequence identity over discrete intervals and encode the same alpha-globin polypeptide. On the other hand, the coding regions of some genes have accumulated replacement changes that result in distinct alpha-globins. In one instance, these changes appear to reflect positive selection of advantageous mutations.

Amino Acid Sequence↗

PubMed1985

The complete primary structure of HLA-Bw58.

Serological studies indicate that HLA-B17 molecules are unusually cross-reactive with products of the HLA-A locus. In particular, a mouse monoclonal antibody MA2.1 defines an epitope that is shared by HLA-A2 and the two subtypes (Bw57 and Bw58) of B17. To investigate these relationships at the structural level, we have isolated a gene coding for Bw58 from the WT49 B cell line. The gene was transfected into mouse L cells and its protein product was characterized with a panel of monoclonal anti-HLA antibodies. The nucleotide sequence of 3520 base pairs of DNA encompassing the seven exons coding for Bw58 and associated introns was determined. The deduced protein sequences for Bw58 and eight other HLA-A,B,C molecules were compared. In the first polymorphic domain (alpha 1), Bw58 is unusual in that it is as homologous to HLA-A locus products as to HLA-B locus products. In the second polymorphic domain (alpha 2), Bw58 has greater homology to B locus products. In the alpha 1 domain of Bw58, small segments of amino acid and nucleotide sequence homology with A2 (residues 62-65) and with Aw24 (residues 75-83) are found in the major region of polymorphic diversity (residues 62-83). These similarities provide structural correlates for the serological relationships between Bw58 and A locus molecules, with residues 62-65 possibly being involved in the MA2.1 epitope. From comparisons of four HLA-A and four HLA-B sequences, there is a difference in the patterns of variation for A and B locus molecules. For B locus molecules there is greater variation in the alpha 1 domain than in the alpha 2 domain. For A locus molecules, variation in the two domains is similar and like that for B locus alpha 2 domains. In comparison to other HLA-A,B,C genes, novel inverted repeat sequences were found in the nucleotide sequence of HLA-Bw58. These sequences flank the putative RNA splicing sites at the 3' end of the exons encoding the alpha 2 and alpha 3 protein domains.

Amino Acid Sequence↗

PubMed1991

Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

Antigens, Bacterial↗

PubMed1991

Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction.

Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.

Base Sequence↗

PubMed2025

Should neighbours of tuberculosis (TB) cases be prioritised for active case finding in high TB-burden settings? A prospective molecular epidemiological study.

INTRODUCTION: In high tuberculosis (TB)-burden countries, considerable transmission of Mycobacterium tuberculosis (M. tb) likely occurs outside of households. We aimed to estimate the TB prevalence and incidence in households and neighbourhoods around known TB cases and to understand transmission patterns. METHODS: Household and neighbourhood contacts of pulmonary TB index cases from contiguous areas in Bandung, Indonesia, were screened and followed up for 12 months. Sputum samples underwent smear microscopy, M. tb culture, Xpert MTB/RIF, DNA isolation and whole-genome sequencing (WGS). Pairwise single-nucleotide polymorphism (SNP) distance ≤12 defined transmission for pairs with known epidemiological links, or SNP≤3 for pairs without epidemiological link. An SNP=12 cut-off was used to characterise transmission clusters. RESULTS: From 213 index cases, 514 household and 4141 neighbourhood contacts underwent TB screening: 19 household (3.70%, 95% CI 2.24 to 5.71) and 45 neighbourhood (1.09%, 95% CI 0.79 to 1.45) contacts were identified with TB, of whom 18 (3.50%, 95% CI 2.20 to 5.48) and 38 (0.92%, 95% CI 0.65 to 1.13) respectively, were bacteriologically confirmed. During follow-up, 11 household and 13 neighbourhood contacts were identified with TB (incidence per 100 000 person-years: 2286 (95% CI 1286 to 4148) and 350 (95% CI 190 to 563)), of whom 6 and 8, respectively, were bacteriologically confirmed (incidence per 100 000 person-years: 1247 (95% CI 560 to 2776) and 201 (95% CI 101 to 402)). A total of 223 patient M. tb isolates underwent WGS. Of 15 intra-household pairs, 8 (53.3%) were transmission pairs. Of 24 neighbour to index case pairs, 1 (4.2%) was a transmission pair. 11 of 19 transmission pairs shared no epidemiological link. We identified 25 M. tb genetic clusters from 205 mono-TB isolates overall. CONCLUSION: Neighbours have lower prevalence and incidence of TB than household contacts, but twice as many cases. Very few received M. tb from their index case, suggesting uncontrolled community-wide transmission. Whole population active case finding may be necessary in high TB-burden settings.

Humans↗

PubMed1992

Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion.

Twenty-seven cases of inflammatory breast cancer were screened for the presence of the p53 protein by immunocytochemical methods using a monoclonal antibody directed against the p53 protein. Three groups were detected: 8 cases (30%) had high levels of p53 in the nucleus of the cancer cells; 9 cases (33%) had a complete lack of detectable staining; 10 cases (37%) showed a pattern of cytoplasmic staining with nuclear sparing. Nucleotide sequence analysis of p53 cDNAs derived from the samples with cytoplasmic staining revealed only wild-type p53 alleles in 6 out of 7 cases. An eighth case was determined to be wild type by a single-strand conformation polymorphism. In contrast, the samples containing nuclear p53 contained a variety of missense mutations and a nonsense mutation. The p53 cDNAs from 3 of the tumors that lacked detectable p53 staining were analyzed, and all 3 had wild-type nucleotide sequences. Interestingly, a case of normal lactating breast tissue also showed intense cytoplasmic staining for p53 with nuclear sparing. These data suggest that some breast cancers that contain the wild-type form of p53 protein may inactivate its tumor-suppressing activity by sequestering this protein in the cytoplasm, away from its site of action in the cell nucleus. The detection of cytoplasmic p53 in normal lactating breast tissue could suggest that this is the mechanism employed in specific physiological situations to permit transient cell proliferation. This observation could explain how some breast cancer tissues inactivate p53 function without mutation.

Amino Acid Sequence↗

PubMed1986

DNA finger printing by oligonucleotide probes specific for simple repeats.

Interspersed simple repetitive DNA is a convenient genetic marker for analysis of restriction fragment length polymorphisms (RFLPs) because of the numbers and the frequencies of its alleles. Oligonucleotide probes specific for variations of the GATCA simple repeats have been designed and hybridized to a panel of human DNAs digested with various restriction enzymes. Numerous RFLPs were demonstrated in AluI and MboI digested DNA with "pure" GATA oligonucleotides as probes. The optimal length of the probe for RFLP analysis was 20 bases taking into account fragment lengths (1.5-7 kilobases = kb), signal to background ratio, and number of clearly evaluable RFLPs. By using different restriction enzymes individual-specific hybridization patterns ("DNA fingerprints") can be established. Hypervariable simple repeat fragments are stably inherited in a Mendelian fashion. Advantages of this method are discussed.

DNA↗

PubMed2026

Fine-Scale Landscape Genomics Show Asymmetric Patterns of Gene Flow for the Invasive Mosquito Aedes albopictus.

Mosquito-borne viruses like dengue, Zika, and chikungunya pose increasing health risks in the United States due to the expanding range of Aedes albopictus, a highly invasive mosquito species that now has a global distribution. Aedes albopictus thrive in artificial containers associated with anthropogenic land use, allowing populations to reach high numbers in urban and suburban environments. While the global spread of Ae. albopictus has been well characterized, the effects of heterogeneous urban landscapes on dispersal and gene flow at fine spatial scales remain unclear. This study analyzed the genetic connectivity of Aedes albopictus populations collected in Wake County, North Carolina in 2018. We used single nucleotide polymorphisms (SNP) data from double-digest restriction-enzyme associated DNA sequencing (ddRADseq) and examined genetic connectivity through principal component analysis (PCA) and genetic network analysis. We then evaluated migration and source-sink dynamics using a Bayesian approach for SNP data (BA3-SNP). We found little evidence of genetic clustering or isolated populations of Ae. albopictus in Wake County, suggesting high gene flow between sites. Migration analysis demonstrated asymmetric gene flow from rural to urban regions within Wake County, with greater gene flow occurring between and within urban regions. These findings suggest that the pattern of gene flow of Ae. albopictus populations within local metropolitan areas may involve urban city centers serving as genetic sinks and surrounding suburban and rural regions serving as sources. This study highlights how heterogeneous landscapes shape mosquito population connectivity and migration at fine spatial scales, which is critical for informing vector control and public health intervention strategies.

Aedes albopictus↗

PubMed2026

Shared candidate genes associated with variation in egg size in cold-adapted and artificially selected Drosophila melanogaster.

The development of most multicellular organisms begins with oogenesis, the production of the egg. In D. melanogaster, egg size is a highly polygenic trait closely related to fitness. Elements of shifts in egg size have been widely studied and modeled, but the genes underlying this variation are still poorly understood. This study aimed to identify candidate genes associated with processes underlying egg-size variation using D. melanogaster as a model. In selection experiments, we generated large-egg populations from a shared ancestral population using both cold-adaptation and artificial selection, and identified candidate genes for the large-egg phenotype. Using whole-genome DNA sequencing and strict computational filtering, we uncovered single-nucleotide polymorphisms in 10 genes. Characterization of these candidates revealed functions in cytoskeletal dynamics, DNA replication and repair, intracellular signaling, and stem cell maintenance and differentiation. RT-PCR and qPCR were used to validate gene expression differences between cold-adapted lines and the Oregon R control (OrR) in a subset of the candidates. In RT-PCR, stathmin demonstrated a modified expression pattern in all cold-adapted lines relative to OrR controls. In qPCR experiments, Pde1c had significantly higher expression (p < 0.05) in the cold-adapted flies compared to OrR controls for all three fly cages tested. For Ino80, significantly higher expression was observed for one of three cages while one cage showed lower expression. We have assembled a candidate list we hope will be a useful resource for researchers across specialties, from germ cells to cytoskeletal dynamics, to further investigate the genetic and developmental aspects of variation in egg size in D. melanogaster.

Animals↗

PubMed1991

Genetic diversity of human parvovirus B19 determined using a set of restriction endonucleases recognizing four or five base pairs and partial nucleotide sequencing: use of sequence variability in virus classification.

Analysis of the restriction site polymorphism (RSP) of human parvovirus B19 using 12 restriction endonucleases (REs) recognizing four or five bp sequences (4- or 5-bp REs) revealed a significant difference between strains previously classified as being of the same genome type, and a relationship between two strains of different genome types, thereby indicating a global spread of B19 virus strains. These findings demonstrate the advantage of this set of 4- and 5-bp REs for the calculation of the degree of genetic diversity and clearly it is necessary to amend the taxonomy of B19 virus strains using these REs. We examined the nucleotide (nt) sequence between nt 3141 and 3411, at the N terminus of the VP2 protein coding region, in 12 B19 virus strains. The pattern of distribution of nucleotide differences between the strains confirmed the classification by RSP analysis. Between nt 3293 and nt 3364, a region in which an antigenic epitope may be encoded, there was no evidence of a nucleotide change causing an amino acid change. Thus, the amino acid sequence in this potential epitope is probably conserved.

Cloning, Molecular↗