Biomedical subjects
Publications and source records attributed to G Lucotte.
Pst I endonuclease fragment length polymorphism of the antithrombin III probe was studied in a French population. Among 25 subjects tested, 15 were found heterozygous at the Pst I site, so that this polymorphism might serve as a useful markers of antithrombin III gene in families with antithrombin III deficiency.
The human Y chromosome carries numerous copies of a tandemly repeated Pvu II sequence, 2.4 kb long. These sequences are specific to humans, and are present in a much smaller amount in the DNA of females. They are localized on the long arm of the Y chromosome. We have compared this sequence with the Hae III 2.1 kb Y-specific repeated sequence, already described.
Hybridization of alpha-fetoprotein clones cDNA with human DNAs digested by seven restriction endonucleases reveals one polymorphism. This polymorphism, detected after restriction by MspI, is at low frequency (f = 0.02) and is validated by family analysis. It corresponds to an intronic sequence, for a methylated site external to the probes utilized.
Restriction enzyme site polymorphisms for Pvu II endonuclease are examined in a panel of DNAs isolated from peripheral blood lymphocytes of HLA-typed individuals. 21 polymorphic bands are detected from a total of 39 restriction fragments analyzed, only 9 of them corresponding to common variable morphs. Three polymorph fragments only correlate with known HLA-A and -B serologic alleles.
Using the Taq I restriction polymorphism of a factor IX probe, we analysed the segregation of this gene and that of the fragile site Xq27. The ancestor of this family was a healthy carrier male. Of twelve informative meioses, at least four recombinations were detected. The hypothesis of a particular instability of the distal part the long arm of the X chromosome is reconsidered.
Restriction polymorphisms of three human genomic probes (RC8, L1-28 and p754), located on the short arm of the X chromosome, are studied in a French population panel after digestion with taq I and Pst I endonucleases. Informativeness of these three probes in that population are, respectively, 0.18, 0.41 and 0.50. Two kinds of strategies for utilizing these polymorphisms to prenatal and carrier diagnosis of the Duchenne muscular dystrophy (DMD) are given. The last probe available, p754, is of great interest, cumulating advantages that being nearly the DMD gene (3 centiMorgans) and presenting the maximal informativeness theoretically possible.
Hybridization of albumin clones cDNA with human DNAs digested by several restriction endonucleases reveals two HaeIII polymorphisms. The first polymorphism, H1, is of low frequency (f1 = 0.05); the second, which is validated by family analysis, occurs frequently (f2 = 0.21) and is an intronic polymorphism, probably of substitution--base type.
Two structural variants of the rat alpha-fetoprotein (AFP) gene have been detected in different inbred strains of rats by EcoRI or HindIII restriction enzyme cleavage of cellular DNA, agarose gel electrophoresis and Southern blot hybridization using 32P-labeled cloned rat AFP cDNA probes. The type I AFP gene variant is characteristic of the Sprague-Dawley strain, and type II is found in Buffalo rats. These variants appear to represent two different allelic forms of the rat AFP gene since they are inherited in a normal Mendelian fashion when Sprague-Dawley and Buffalo rats are crossed. The mapping results suggest that the two allelic variants differ from each other by multiple cleavage site variations (base pair substitutions) and by an insertion or deletion of DNA sequences. An extensive sequence variation appears to exist between the two forms of the rat AFP gene; we have estimated that as much as 2.7% of the nucleotides in this region vary between the two alleles.
Restriction fragment length polymorphism of the RC8 probe, which is loosely linked to the Duchenne muscular dystrophy locus, was studied in a French population. Among 22 females, 18.1% were found to be heterozygous for the two frequent B1 and B2 alleles, and a rare allele was found in 1 woman with a corresponding variant band at 3 kb. Among 18 males, 6 were found to have the B2 allele. The B2 gene frequencies were 0.09 and 0.33 in males and females, respectively. This difference was statistically significant (p less than 0.05), but may nevertheless be fortuitous. There was no significant gene frequency difference between the English and French populations.
An association was found between idiopathic haemochromatosis and a 6.2-kb Bgl I genomic DNA fragment. This fragment constitutes a class I sequence which correlates with HLA-B3. The Bgl I fragment could represent a sequence in linkage disequilibrium with the haemochromatosis gene.
After electrophoresis and transfer onto a hybridization membrane, the restriction endonuclease fragments obtained were probed with a cDNA carrying the nucleotide sequence encoding a class I HLA gene. Polymorphism for presence or absence of various Eco RI and Eco RV and Hind III fragments was noted in a panel of unrelated Parisian people: frequent polymorphism was described for 2 Eco RI restriction fragments of sizes 16.5 and 7.7 kb, for 5 Eco RV restriction fragments of sizes 25, 20, 11.5, 7.8 and 4 kb, and for 3 Hind III restriction fragments of sizes 27.5, 23.5 and 4.7 kb. The French population studied is in Hardy-Weinberg equilibrium for the different variants described.
The possibility that a plasmid carried by Klebsiella pneumoniae plays a role in the pathogenesis of ankylosing spondylitis was explored. K. pneumoniae K21 contains a congruent to 25-kb plasmid, but this plasmid is not present in lymphocyte DNAs of ankylosing spondylitis HLA-B27 patients, as demonstrated by molecular hybridization experiments.
Among the different methods for isolation of high-molecular-weight DNA from leucocytes, two of them were retained as being the most interesting and were compared. This choice was based on three criteria: the quality of the DNA obtained (high-molecular-weight DNA, RNA, and protein free), the efficiency of the method (in terms of the yield of DNA obtained), and the ease and length of the method. The first method described was chosen as a reference (100% efficiency); in comparison, the second one had a 70% efficiency, but was shorter and easier to handle.
Numerous biological factors have an effect upon the probability of the presence of a theoretical restriction site at a given point, (1/4)n, in a DNA molecule, where n represents the number of base pairs composing the site. Indeed, it is a fact that the ratio (formula; see text) does not usually equal unity. In addition, the sequence of the nucleotides appears to change haphazardly, with certain dinucleotides present more often than others. When considering (formula; see text), it is observed that the influence of this first parameter is very important for sites with either four or six base pairs. The balance of the average frequency of each site according to the preponderance of certain dinucleotides and the G + C content has been the subject of an involved study which makes use of a certain number of restriction enzymes which are commercially available. The important frequency variations of different sites are due primarily to the presence of one or two GC doublets which go as far as reducing the frequency by a factor of 3.
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High molecular weight DNA extracted from 50 unrelated parisian individuals constitutes a "DNA-thèque" which can be used at the population level. This procedure allows preparation from a variety of tissues such as liver, placenta and leucocytes. The "quality" of the DNA obtained (about 50 Kb, RNA and protein free) and the efficiency of the method (in terms of the yield of DNA obtained) are considered. Blood is the easiest material to obtain and permits the obtaining of a reasonable amount of human and primate DNA, particularly when familial investigation is planned for polymorphism studies. One of the objects of this article is to encourage others to make use of our bank, for studies in population and evolutionary genetics.
Variations were observed at the prealbumin-2, carbonic anhydrase-I, 6-phosphogluconate dehydrogenase, phosphoglucomutase-II and adenylate kinase loci in Erythrocebus patas subspecies pyrrhonotus. But the level of heterozygosity was very low compared with those of the different African monkey species studied by the same electrophoretic analysis. The reasons for this low variation are discussed with regard to ethological and genetic structure characteristics of the group studied.
The techniques involved in the measurement of the electrophoretic mobilities of human proteins have been applied for similar protein electrophoretic mobility measurements in various species of anthropoïd apes. A study of the electrophoretic polymorphism in the chimpanzee shows that this species has a similar variability to that of man, although the average individual heterozygosity level is lower. For a certain number of erythrocyte protein loci, the electrophoretic mobilities in man, chimpanzee, and gorilla are about the same. The two types of chimpanzee are distinguished by their distinct allelic forms at the level of only one locus, the transferrin locus. The various sub-species of the common chimpanzee, gorilla, and orang-utan differ at the discriminative post-albumin and transferrin locus, not only in their allelic forms but also in the degree of heterozygoty. A study of such differences enables the proposal of a hypothesis about the relationships between these diverse races. Amongst these, the two sub-species of orang-utan are distinguishable by other diagnostic markers. It would seem that in a study of specific differentiation applicable to that of the human species, the different types of baboons constitute a better model than that of the various anthropoïd apes.