Construction and sequence analysis of subtraction complementary DNA libraries from human preimplantation embryos.

PURPOSE: Because stage-specific genetic expression in human preimplantation development is not sufficiently studied, we have undertaken the construction of a subtraction complementary DNA (cDNA) library enriched for transcripts specific for human blastocysts. METHODS: For this purpose individual pools of cDNAs synthesized from four hatched blastocysts and three cleaving 8- to 10-cell embryos were exposed to suppression subtractive hybridization to minimize the presence of transcripts of housekeeping genes and other genes of maternal origin known to be expressed earlier in preimplantation development. Random clones of this library were sequenced and analyzed using the BLAST algorithm. RESULTS: The resulting subtraction library had a complexity of 3 x 10(5) and an average size of inserts of about 0.8 kb. Sequencing of random library clones revealed the following human genes: CD9 antigen, fatty acid binding protein, ferritin heavy chain, amyloid precursor, MAP kinase messenger RNAs, DNA clone 127H14, messenger RNA for diacylglycerol kinase, a sequence homologous to C1 inhibitor, messenger RNA for the KIAA0145 gene, and others. CONCLUSIONS: The presence of these genes in human preimplantation development suggests expression specific to the blastocyst stage.

Isolation of cDNA libraries from individual human preimplantation embryos.

Although available data from the mouse model suggest that morphogenesis during preimplantation development is dependent on the expression of embryonic genes, convincing data for human preimplantation embryos are missing. To investigate the expression of genes involved in human preimplantation development we constructed cDNA libraries from human individual blastocysts and screened them for the expression of beta-actin, CD59, homeoboxes OCT-3 and HOXA4, and HLA-G and hMLH-1 genes. Beta-actin, CD59, and OCT-3 were detected by reverse transcription-polymerase chain reaction (RT-PCR), while HOXA4, HLA-G and hMLH-1 were undetected. Sequencing of 48 random clones from two libraries revealed a different identity to the known genes including 99% identity to human histone 3.1 and human ribosome protein S25 complete cDNA. These data demonstrate the feasibility of constructing cDNA libraries from individual human preimplantation embryos and their potential usefulness in the assessment of the relevance of specific gene expression in the failures of preimplantation development.

Sequence analysis of libraries from individual human blastocysts.

PURPOSE: It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of beta-actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. METHODS: cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. RESULTS: The presence of two housekeeping genes, hexokinase I and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. CONCLUSIONS: The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their values in the analysis of genetic expression in human preimplantation development.

Evolutionary classification of homeodomains.

PURPOSE AND METHODS: We performed multiple comparisons between available amino acid (aa) sequences of homeodomain(HOM)-containing proteins from a wide spectrum of animals to create an evolutionary classification of the proteins. RESULTS: Based on results of statistical and special computational analyses of over 500 homeodomain aa sequences (HOMs) a novel system of concepts describing complex structural correlations between homologous proteins is proposed. This system includes such notions as differentiated isofunctionality of aa, chemotype, stereotype, local functional motifs, gradual conservativeness of aa positions, and group-specific domain patterns, as well as major categories of the evolutionary classification of HOMs (Division, Type, Branch, Class, Family, Series, Variety, Sort). Using this approach, a complete structural systematics of HOMs belonging to proteomes of eukaryotic animals is proposed. CONCLUSIONS: The proposed structural classification of HOMs is in full agreement with the bulk of experimental data revealing complex functional similarities and differences among HOMs in terms of their expression patterns in developing embryos. It turn, this classification can provide answers regarding homology among homeodomains when experimental data are conflicting.

Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies.

PURPOSE: The purpose of the study was to investigate homeobox gene expression in human oocytes and preembryos and in postimplantation embryos with impaired embryonic development determined by chromosomal abnormalities. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets for Homeobox gene sequences was used. RESULTS: The homeobox genes HoxA4, HoxA7, HoxB4, and HoxB5 were present in human oocytes and cleaving normal and triploid embryos. The expression pattern was different between chromosomally abnormal and normal first-trimester embryos. Of four homeobox transcripts (HoxA7, HoxB4+ ++, HoxB5, and HoxC6) that are expressed in diploid embryos, only HoxA7, HoxB4 and HoxC6 were present in a trisomy 7 embryo, and only HoxB4 and HoxB 5 in triploid embryos and an embryo with trisomy 9. Cloning experiments revealed differences in the number of homeobox clones obtained from trisomy 7 and control embryos. CONCLUSIONS: The transcripts of homeobox genes, HoxA4, HoxA7, HoxB4, and HoxB5, were present in oocytes and cleaving embryos. The pattern of expression of homeobox genes in cultured fibroblasts derived from spontaneously aborted embryos with aneuploidies was different from that in control diploid cells.

Homeobox gene expression in human oocytes and preembryos.

Homeobox gene expression in human preimplantation development has not been established. We used reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets to investigate the presence of mRNA of homeobox genes in human oocytes and preembryos. RT-PCR products obtained from normal and unfertilized oocytes, from cleaving normal and triploid embryos, and from morulae and blastocysts were cloned, sequenced, and analyzed for the presence of homeobox sequences. The presence of mRNA of homeoboxes HoxA4 and HoxA7 was demonstrated; HoxA4 was present in normal and unfertilized oocytes and also in a 4-cell embryo. HoxA7 was present in normal oocytes and cleaving triploid embryos.

Review: borders, patterns, and distinctive families of homeodomains.

PURPOSE: Homeotic proteins function as transcription factors in early embryogenesis of many organisms. To date, hundreds of distinctive homeoproteins have been identified, including 84 human homeodomains. However further progress in understanding functional relationships between particular homeoproteins and other embryonic regulators requires a comprehensive structural classification of these proteins. RESULTS: The most probable borders and conservative amino acid positions inside the homeodomain region have been established using a statistical analysis of variabilities of amino acid occurrences at various positions outside and inside the domain. A new format for a homeodomain sequence presentation and regular amino acid patterns which are strongly representative of distinctive homeodomain groups are proposed. Using the established patterns, 33 families of closely related homeodomains have been distinguished and classified. The total list of 297 homeodomain amino acid sequences is presented in the Appendix. CONCLUSION: The structural classification of homeodomains has been proposed. It can be useful for both the identification (or prediction) of new homeotic genes/proteins and the recognition of possible PCR-induced sequence errors. This systematics will also have an impact on understanding functional relationships among homeotic proteins and other genetic regulators of developmental processes.

[In situ hybridization in human genome mapping]. / Gibridizatsiia in situ v reshenii problemy kartirovaniia genoma cheloveka.

The methodological possibilities of non-radioactive in situ hybridization of nucleic acids and its application for an immediate localization of genes and chromosomes are discussed. The advantages of a non-isotope probe labeling versus a use of radioactive substances are emphasized. Various types of compounds used as a label are distinguished. The principles of use of the above labels and the ways to improve the method in terms of increasing its sensitivity are considered. Some results obtained while using non-radioactive labelled probes are reported. It is shown promising to use this method for molecular-genetic analysis of DNA polymorphism in human genome.

[Study of polymorphism in human rRNA gene clusters. Population and familial analysis of basic types of RFLP]. / Issledovanie polimorfizma v predelakh klaster genov rRNK cheloveka. populiatsionnyi i semeinyi analiz osnovnykh tipov PDRF.

The frequency and average amount of copy number per genome were defined for standard and a number of new variants of BamHI 5'-NTS RFLP from populations of Moscow, Riga and individuals with Down syndrome. It was demonstrated that the populations studied differ neither in population frequency nor in the average amount of copy number of the variants. New variants were detected in the EcoRI 3'-NTS RFLP system and their amplification, as well as discordance among MZ twins. Possible target for methylation in the HindII site of 3' end of 28S rRNA gene was revealed. Analysis of data obtained demonstrated inefficiency of using the RFLP systems in systematic mapping of NOR-chromosomes. Our data also suggested a possible role of amplification of one copy repeated unit rRNA genes in their evolution.

[Study of polymorphism in human rRNA gene clusters]. / Issledovanie polimorfizma v predelakh klastera genov rRNK cheloveka.

Human ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene was used for detecting standard restriction fragments' length polymorphism (RFLPs) in the non-transcribed spacer. The conditions for hybridization of rDNA probe which eliminate cross hybridization of parts of 28S rRNA gene were developed. A test for detecting incompletely restricted DNA was also developed which may be used in experiments for detecting new RFLPs. It was found that a set of standard RFLPs was identical in various human tissues for one individual. Frequency of standard RFLPs in the non-transcribed spacer of human rRNA gene clusters was calculated.

[Genetico-demographic patterns of the prevalence of various forms of endogenous psychoses]. / Genetiko-demograficheskie zakonomernosti rasprostraneniia razlichnykh form éndogennykh psikhozov.

Prevalence rates (PRs) for EFP (schizophrenic, schizoaffective and affective psychoses), with allowance for proband sex and age-of-onset data were studied in a subdivided population from the North-East of the European Region of the USSR. The population includes three subpopulations: a small old religious semi-isolate of Russians ("Rs"), aboriginal Komi people ("Ks")--an ethnic community of Ugro-Finnish lineage, and a mixed group of migrants ("Ms") from various regions of the USSR. The latter is mainly an urban population, while the "Rs" and "Ks" are, on the whole, rural populations. The total PR for EFP was found to be 0.97% for the "Rs", 0.63% for the "Ks" and 0.35% for the "Ms", whereas PRs-0.85-1.15% in other parts of the USSR, mainly for "panmixed" populations in large towns. The lower PRs for EFP in the "Ms" is caused by a backmigration flow involving certain groups of patients; consequently, the mean liability for "Ms" offsprings (as a whole) should also be lower. On the other hand, the lower PRs for EFP in the "Ks" is caused by underpresentation of clinically mild cases of the mental disease (mainly, pseudoneurotic schizophrenia), especially among female patients, probably due to that the so affected persons are sufficiently adapted to the cultural traditions of this rural population. It was shown that in the "Rs" the total PR for "nuclear" and paranoid schizophrenia is 0.68% versus 0.25% in a "panmixed" population. The increase is most likely caused by the high inbreeding level in the "Rs" semi-isolate, and if this is correct, we may suppose that at least one or two recessive genes are involved in the liability to the most heavy forms of schizophrenia. On the other hand, in the "Ms" (as in other "panmixed" populations) positive assortative mating among hereditary-predisposed persons is a more significant factor influencing family transmission of EFP, since the correlation between probands and their spouses is rpp = 0.31 (p less than 0.001) in the "Ms", as compared to rpp = 0.19 (p less than 0.1) in the "Rs". Thus, our general conclusion is that neither the place of inhabitance nor the life mode are the causal factors for EFP, but rather some genetic factors, more accurately, certain sets of specific genes.

[A method of analyzing the chromosome localization of repetitive nucleotide sequences by using hybridization in situ]. / K metodike analiza khromosomnoi lokalizatsii povtoriaiushchikhsia nukleotidnykh posledovatel'nostei s pomoshch'iu gibridizatsii in situ.

To increase precision in the quantitative analysis of chromosomal distribution of repeated DNA sequences detected by in situ hybridization, a number of factors important in studies of the molecular basis of chromosome polymorphism should be considered. While analysing results of hybridization, one should only use the number of grains over the sites of their regular localization, instead of those over the whole chromosome. It is also evident that to decrease variability conditioned by differences in the labelling degree of separate cells, the relative numbers of labelled grains over chromosomes should be used in the analysis and not the absolute ones. Data from those cells ought to be only included in the analysis, in which the labelling degree is sufficient to show all localization sites of the repeated sequences studied, i.e. cells should be used with comparatively constant relative numbers of labelled grains over all their regular localization sites.

[Genetic analysis of predisposition to cancer of the stomach]. / Geneticheskii analiz predraspolozheniia k raku zheludka.

The results of a complex family and population epidemiologic study of gastric cancer pointed to inheritance as an important factor of the incidence of this disease. However, there may be different combinations of genetic factors, on the one hand, and genetic and environmental ones, on the other, versus age and sex. This should be considered in conducting screening for families at genetic risk for stomach cancer and taking measures aimed at eliminating carcinogenic factors which increase the likelihood of familial cancer incidence. Such considerations should contribute to a certain degree to early diagnosis and prevention of the disease.

[Clinico-genetical studies of colonic cancer. III. Primary multiple malignant neoplasms]. / Kliniko-geneticheskie issledovaniia raka tolstoi kishki. Soobshchenie III, Pervichno-mnozhestvennye zlokachestvennye novoobrazovaniia (PMZN).

The results of clinico-genealogic analysis of 46 patients with primary-multiple malignant neoplasms are given (among them 16 patients with primary-multiple malignant neoplasms of colon cancer and 30 patients with one or more neoplasms in combination with different malignant tumors of other organs). The values of segregation rates obtained for primary-multiple malignant neoplasms are lower than theoretically expected for simple monogeneous types of inheritance. The relation analysis of primary-multiple malignant neoplasms and colon cancer revealed that these tumors are likely to appear among relatives of probands under the influence of the same genetic system of determination. Risk of the colon cancer development for relatives of the patients with primary-multiple malignant neoplasms is higher than for relatives of the patients with colon cancer.

[Clinico-genetical studies of colonic cancer. II. Genetic analysis of predisposition to colonic cancer]. / Kliniko-geneticheskie issledovaniia raka tolstoi kishki. Soobshchenie II. Geneticheskii analiz predraspolozheniia k raku tolstoi kishki.

The structure of subjection to different clinical forms of colon cancer and to the morbidity as a whole approximates better the quasi-continued phenotypical model within which the contribution of genetic factors reaches 68-84%, that of incidental medium factors being 16-32%. Genetic study of heterogeneity of colon cancer clinical forms revealed that their pathogenetic community was quite high. However, the origin of colon cancer depends strongly on genetic factors (83.7 +/- 7.3%), in comparison with rectal cancer (67.9 +/- 7.1%). The analysis of colon cancer interrelation with other malignant neoplasms (including specific ones for women--breast and uterus cancer) revealed that the development of another malignant neoplasms was the result of the influence of partially common genes (20-50%) which predetermined the development of colon cancer and other malignant neoplasms. According to the data obtained in this study, the tables of repeated risk have been worked out which may be used for medico-genetic consultation.

[Clinico-genetical analysis of colonic cancer. I. Occurrence, frequency in families and segregation analysis]. / Kliniko-geneticheskoe issledovanie raka tolstoi kishki. Soobshchenie I. Rasprostranennost', semeinoe nakoplenie i segregatsionnyi analiz.

The data on clinico-genealogic studies of colon cancer are presented. 694 families were examined with 432 probands having rectal and 262 colonic carcinoma among them. Clear family accumulation of colon cancer (2.4 +/- 0.35%) as well as other malignant tumors (6.8 +/- 0.6%) (p less than 0.01) was shown among the relatives of the first degree of relation. The values of segregation rates obtained for clinical forms of colon cancer were lower than theoretically expected for simple monogenic types of inheritance. The analysis of incomplete penetration of genotypes showed that, though formally the inheritance of colon cancer and its clinico-anatomical forms may be described by quasi-dominant types of inheritance, the penetration values are very low: from 4.3 to 13.3% for homozygotes and from 2.1 to 6.6% for heterozygotes. It shows that the supposition about the monogenic types of the colon cancer inheritance is doubtful and suggests that the colon cancer is to be regarded on the basis of the multifactorial model.

[Cloned fragment of human alphoid DNA--a molecular marker of the pericentromeric region of chromosome 18]. / Klonirovannyi fragment al'foidnoi DNK cheloveka--molekuliarnyi marker pritsentromernogo raiona 18-i khromosomy.

From the library of cloned fragments of human DNA we have isolated two recombinant plasmids containing alphoid DNA sequences pBRHS13, pBRHS65. Both cloned sequences hybridized in situ predominantly to pericentromeric regions of chromosome 18 and with less intensity to pericentromeric regions of chromosomes 2, 9, 20, and were characterized by populational copy number polymorphism in homologous chromosomes. These sequences may appear very useful in the diagnostics and cytogenetic analysis of chromosomal aberrations and in studies of polymorphisms of heterochromatic regions of human chromosomes.