Study of alpha-thalassemia in northeastern Thailand at the DNA level

The frequency of alpha-thalassemias in a general population sample from northeastern Thailand and in an Austroasiatic group with high frequencies of hemoglobin E and beta-thalassemia, the So, was estimated using DNA techniques. Among 64 healthy adult subjects from the Khonkaen and Ubol areas, the following haplotype frequencies were determined: alpha alpha, 0.742; -alpha 3.7 (subtype I), 0.148; -alpha 4.2, 0.016; -alpha del, 0.008; alpha Constant Spring alpha, 0.055; --SEA, 0.023, and alpha alpha alpha (triplicated alpha-globin gene), 0.008. In the So group, the combined frequency of alpha-thalassemia chromosomes was 0.525.     

Human mitochondrial DNA types in two Israeli populations--a comparative study at the DNA level.

Variations in human mtDNA restriction endonuclease fragment patterns were investigated in a sample number of 81 Israelis--Jews and Arabs--using total blood cell DNA. Eight new morphs were observed using five enzymes: HpaI, BamHI, HaeII, MspI, and AvaII. Of the 18 different combinations of fragment patterns (mtDNA types), only three were shared by both groups, but with striking frequency differences. The Arab sample disclosed "African" characteristics and was found to be slightly more polymorphic than the Israeli sample. One of the new types filled a "missing link" originally postulated in a phylogeny of mtDNA human types.     

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins. Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication. Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process. Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis. A possible role of the maturation promoting factor (MPF) is suggested.     

Fronto-ethmoidal encephalomeningoceles in the population of northern Thailand

Summary Fronto-ethmoidal encephalomeningoceles are rare in europoid populations, but seem to be common in Thailand. In a population survey in a limited area in northern Thailand an incidence of 9 cases in a total population of 31,582, i.e. approximately 1:3,500 was found. Studies in 14 families with an affected member indicate that there is no simple Mendelian inheritance, but a contribution of genetic factors to the etiology of this type of fronto-ethmoidal encephalomeningocele is suggested by the occurrence of the defect in 4 close relatives of affected subjects.

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Zusammenfassung Fronto-ethmoidale Encephalomeningocelen sind in europiden Populationen selten, scheinen jedoch in Thailand häufig zu sein. Bei einer Bevölkerungserhebung in einem begrenzten Bereich in Nord-Thailand fanden sich 9 Fälle in einer Population von 31582, d.h. ca. 1:3500. Untersuchungen in 14 Familien mit einem befallenen Probanden zeigten, daß kein einfacher Mendelscher Erbgang vorliegt; jedoch wird eine Beteiligung einer genetischen Disposition für diesen Typ fronto-ethmoidaler Encephalomeningocelen dadurch nahegelegt, daß die gleiche Anomalie bei 4 nahen Verwandten von Probanden gefunden wurde.

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On leave from Universitäts-Kinderklinik Bonn.

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Established and supported by Stiftung Volkswagenwerk, Hannover.     

Prevalence of α-thalassemias in northern Thailand

The population of northern Thailand has one of the highest frequencies of α-thalassemia in the world. However, the available distributional data are controversial. In addition to deletional types of α-thalassemia Hb, type Constant Spring should also be taken into consideration in α-thalassemia population studies, because it causes clinical α-thalassemia in the homozygous state or when present with both α-globin genes deleted in trans. We have examined a sample of 215 healthy subjects from four rural districts of Chiang Mai province. Out of these, 77 exhibited anomalies of the α-globin genes (αα/-α^3.7 in 36; -α^3.7/-α^3.7 in 3; ––^SEA in 30; αα/α^CSα in 5; ααα^anti3.7 in 3). Therefore, no fewer than 2% of the children in northern Thailand are expected to be born with HbH disease or thalassemic hydrops fetalis. The considerable public health problem of hemoglobinopaties and the increasing acceptance of family planning necessitates facilities for the pre- and postnatal diagnosis of these disorders at the DNA level. Received: 4 March 1996 / Revised: 9 April 1996     

DNA polymerase activity at the single-molecule level

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3'→5' exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase-DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA-protein complexes.     

Efficacy of Esbiothrin mosquito coils at community level in northern Tanzania

Field trials with mosquito coils containing 0.15% Esbiothrin at Usa River settlement, northern Tanzania, during 1986 achieved a 52% reduction in biting rates of Anopheles arabiensis Patton and a 73% reduction of Culex quinquefasciatus Say mosquitoes. No mosquito biting activity occurred during the coil burning period which started at 20.00 hours daily and lasted for an average of 8.6 h. A sociological survey indicated that the coils were favourably accepted and produced no serious side-effects.     

Patterns of child fosterage in rural northern Thailand

Evolutionary theory guides an investigation of foster parent selection in two northern Thai villages with different biosocial environments: one village has high levels of labour migration and divorce, and growing numbers of parental death due to HIV/AIDS, while the other village has lower migration, divorce and parental mortality levels. Focus groups examine mothers' and fathers' motivations and ideals regarding foster caretaker selection, and quantitative family surveys examine real fostering outcomes: specifically, the laterality (matrilateral versus patrilateral) and genetic distance of the foster caretakers of all ever-fostered children in these two villages. As predicted, in environments of high marital stability and paternity certainty, parents seem to prefer close genetic kin from either side as foster parents for their children. In low marital stability and paternity certainty environments, parents trust their own lateral kin, regardless of genetic distance, over close genetic kin from the other side. The striking exception to this pattern, however, occurs in the case of parental death, in which case children are fostered to the deceased parent's kin, regardless of the child's sex or other factors. In general, the foster parents for girls are selected with more care, reflecting the daughter/female preference expected in traditionally matrilineal, matrilocal societies. An ordered decision-making pathway for foster parent selection is proposed, taking into consideration the key factors of (a) the circumstances driving the fostering decision, (b) the gender of the child, (c) the gender of the key decision-making parent, and (d) the degree of marital and population fluidity (and subsequent, paternity certainty) in the village.     

Aeroallergens in northern and southern provinces of Thailand

An aeroallergen survey in two provinces, Chiangmai in the northern and Songkhla in the southern part of Thailand which are 1 500 km apart, was done in 1986 and 1987 using a rotorod sampler. The five most common pollen types and mold spores were: Chiangmai: Mold spores: Cladosporium, rust, Fusarium, Nigrospora, 1-celled spore. Pollen grains: Mimosa, wild grasses, Urticaceae, Cyperaceae, tetrad pollen. Songkhla: Mold spores: Cladosporium, rust, Nigrospora, Papularia, 1-celled spore. Pollen grains: Casuarina, Mimosa, Urticaceae, Cyperaceae, wild grass. There were definite differences in the amount and frequency of occurrences of aeroallergens between these two provinces. Furthermore, some specific pollen and mold spores, such as tetrads and Mimosa pollen, were found only in Chiangmai, and Casuarina pollen and Papularia spores only in Songkhla. These findings might be caused by the differences in geography, plantation and meteorological data.     

Identification of bovine K-casein genotypes at the DNA level

By using a bovine kappa-Cn cDNA as probe and the PstI endonuclease we demonstrate that the DNA restriction patterns of kappa-Cn AA and kappa-Cn BB cows are different. Besides two invariant fragments (about 6.8kb and 1.1kb) the former shows two fragments of about 4.3 kb and 0.3 kb and the latter one fragment of about 4.6 kb. kappa-Cn AB cows show intermediate pattern. Therefore, it is possible to determine the bovine kappa-Cn genotypes even in absence of gene product.     

Visualizing protein movement on DNA at the single-molecule level using DNA curtains

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.     

Analysis of plasmid deletional instability in Bacillus subtilis.

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.     

Visualizing helicases unwinding DNA at the single molecule level

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.