Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study.

OBJECTIVE: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol. METHODS: (i) Group without lesions. Smears obtained from the lower lip, border of the tongue and floor of the mouth of 31 control individuals (group I), 49 tobacco users (group II) and 27 tobacco/alcohol users (group III) were stained using the Papanicolaou method. The first 100 cells counted on each smear determined the maturation pattern and the keratinization index (KI). Analysis of variance (ANOVA) and the Tukey multiple comparison test were used for statistical analysis, at a 5% significance level. (ii) Group with lesions. Cytopathological and histopathological studies were conducted for 15 patients: eight with leucoplakia without epithelial dysplasia, two with epithelial dysplasia and five with squamous cell carcinoma. RESULTS: (i) Group without lesions. Statistical analysis revealed a smaller number of superficial cells with nuclei in all sites of the group of tobacco/alcohol users (group III) when compared to the control group (group I), and this difference was statistically significant (P<0.005). (ii) Group with lesions. The severity of histopathological findings increased with the increase in the number of cells of the deeper epithelial layers, with a statistically significant difference in the number of intermediate (P=0.013) and parabasal cells (P=0.049), which increased with the severity of the epithelial maturation disorder: leucoplakias with dysplasia had a greater number of intermediate and parabasal cells than leucoplakias without dysplasia; and the number in squamous cell carcinomas was greater than in leucoplakias with dysplasia. CONCLUSION: The maturation pattern of cells in the three anatomic sites showed changes that may be associated with the synergistic effect of tobacco and alcohol. Also, the severity of histopathological findings was associated with the increase in the number of cells in the deeper epithelial layers.

Physical localization and order of genes in the class I region of the bovine MHC.

Fluorescence in situ hybridization (FISH) analyses were used to order 16 bacterial artificial chromosomes (BAC) clones containing loci from the bovine lymphocyte antigen (BoLA) class I and III regions of bovine chromosome 23 (BTA23). Fourteen of these BACs were assigned to chromosomal band locations of mitotic and pachytene chromosomes by single- and dual-colour FISH. Dual-colour FISH confirmed that class II DYA is proximal to and separated from BoLA class I genes by approximately three chromosome bands. The FISH results showed that tumour necrosis factor alpha (TNFA), heat shock protein 70 (HSP70.1) and 21 steroid dehydrogenase (CYP21) are closely linked in the region of BTA23 band 22 along with BoLA class I genes, and that male enhanced antigen (MEA) mapped between DYA and the CYP21/TNFA/HSP70.1 gene region. All BAC clones containing BoLA class I genes mapped distal to CYP21/TNFA/HSP70.1 and centromeric to prolactin (PRL). Myelin oligodendrocyte glycoprotein (MOG) was shown to be imbedded within the BoLA class I gene cluster. The cytogenetic data confirmed that the disrupted distribution of BoLA genes is most likely the result of a single large chromosomal inversion. Similar FISH results were obtained when BoLA DYA and class I BAC clones were mapped to discrete chromosomal locations on the BTA homologue in white-tailed deer, suggesting that this chromosomal inversion predates divergence of the advanced ruminant families from a common ancestor.

Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase alpha and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat.

The enzyme acetyl-CoA carboxylase alpha (ACC-alpha) is rate-limiting for the synthesis of long-chain fatty acids de novo. As a first characterization of the bovine gene encoding this enzyme, we established the entire bovine ACC-alpha cDNA sequence (7041 bp) and used experiments with 5' rapid amplification of cDNA ends to determine the heterogeneous composition of 5' untranslated regions, as expressed from three different promoters (PI, PII and PIII). The individual locations of these promoters have been defined within an area comprising 35 kbp on Bos taurus chromosome 19 ('BTA19'), together with the segmentation of the first 14 exons. Primer extension analyses reveal that the nutritionally regulated PI initiates transcription from at least four sites. PI transcripts are much more abundant in adipose and mammary-gland tissues than in liver or lung. A 2.6 kb promoter fragment drives the expression of reporter genes only weakly in different model cells, irrespective of stimulation with insulin or dexamethasone. Thus bovine PI is basically repressed, like its analogue from rat. Finely graded deletions of PI map two separate elements, which have to be present together in cis to repress bovine PI. The distal component resides within a well-preserved Art2 retroposon element. Thus sequence, structure and evolutionary origin of the main repressor of PI in bovines are entirely different from its functional counterpart in rat, which had been identified as a (CA)(28) microsatellite. We show that, in different mammalian species, unrelated genome segments of different origins have been recruited to express as functionally homologous PI the ancient and otherwise highly conserved ACC-alpha-encoding gene.

Mining the mammalian genome for artiodactyl systematics.

A total of 7,806 nucleotide positions derived from one mitochondrial and eight nuclear DNA segments were used to provide a robust phylogeny for members of the order Artiodactyla. Twenty-four artiodactyl and two cetacean species were included, and the horse (order Perissodactyla) was used as the outgroup. Limited rate heterogeneity was observed among the nuclear genes. The partition homogeneity tests indicated no conflicting signal among the nuclear genes fragments, so the sequence data were analyzed together and as separate loci. Analyses based on the individual nuclear DNA fragments and on 34 unique indels all produced phylogenies largely congruent with the topology from the combined data set. In sharp contrast to the nuclear DNA data, the mtDNA cytochrome b sequence data showed high levels of homoplasy, failed to produce a robust phylogeny, and were remarkably sensitive to taxon sampling. The nuclear DNA data clearly support the paraphyletic nature of the Artiodactyla. Additionally, the family Suidae is diphyletic, and the nonruminating pigs and peccaries (Suiformes) were the most basal cetartiodactyl group. The morphologically derived Ruminantia was always monophyletic; within this group, all taxa with paired bony structures on their skulls clustered together. The nuclear DNA data suggest that the Antilocaprinae account for a unique evolutionary lineage, the Cervidae and Bovidae are sister taxa, and the Giraffidae are more primitive.

Genetic analyses of two independent chlorophyll-deficient mutants identified among the progeny of a single chimeric foliage soybean plant.

Chimeric (variegated) foliage plants are frequently observed in many species. In soybean [Glycine max(L.) Merr.], progeny of chimeric plants are a source of nuclear and cytoplasmically inherited mutants. Self-pollinated progeny of a single chimeric plant derived from tissue culture of PI 427099 (Jilin 3) included plants with green foliage, chimeric foliage, yellow foliage (viable), and yellow foliage (lethal). Our objectives were to determine (1) inheritance, linkage, and allelism of the lethal-yellow mutant with known chlorophyll-deficient mutants; (2) inheritance, linkage, and allelism of the viable-yellow mutant with known chlorophyll-deficient mutants; (3) allelism of the lethal-yellow mutant with the viable-yellow mutant; and (4) male and female gamete transmission of the viable-yellow mutant trait. The viable-yellow mutant was allelic to T323, y20 y20 (Ames 2) Mdh1-n Mdh1-n (Ames 2) and was assigned genetic type collection number T361 and gene symbol y20 y20 (Ames 24) Mdh1-n Mdh1-n (Ames 22). The lethal-yellow mutant was allelic to T225H (Y18 y18) and was assigned genetic type collection number T362H and gene symbol Y18 y18 (Ames 2). T225H became Y18 y18 (Ames 1). The two chlorophyll-deficient mutants were not linked to each other. There was no significant difference in F(1) male or female gamete transmission of the viable-yellow mutant. However, many cross-combinations gave significant deviations from the expected 3 green plants:1 viable-yellow plant in the F(2) generation. The allelism of these two chlorophyll-deficient mutants with mutants T225H and T323, derived from putative transposable element systems, is intriguing. An explanation of this phenomenon awaits molecular experimentation.

A molecular cytogenetic analysis of the tribe Bovini (Artiodactyla: Bovidae: Bovinae) with an emphasis on sex chromosome morphology and NOR distribution.

Q-band comparisons were made among representative species of the four genera of the tribe Bovini (Bos, Bison, Bubalus, Syncerus) as well as to selected outgroup taxa representing the remaining two tribes of the subfamily Bovinae (nilgai, Boselaphini; eland, Tragelphini), the Bovidae subfamily Caprinae (domestic sheep) and the family Cervidae (sika deer and white-tailed deer). Extensive autosomal arm homologies were noted, but relatively few derivative character states were shared. Focus was then made on variation of the sex chromosomes and the chromosomal distribution of nucleolar organizer regions (NORs). Bovine BAC clones were used in molecular cytogenetic analyses to decipher rearrangements of the sex chromosomes, and a pocket gopher 28s ribosomal probe was used to map the chromosomal locations of nucleolar organizing regions (NORs). Some of the more noteworthy conclusions drawn from the comparative analysis were that: 1. The Bovidae ancestral X chromosome was probably acrocentric and similar to acrocentric X chromosomes of the Bovinae; 2. The domestic sheep acrocentric X is probably a derivative character state that unites non-Bovinae subfamilies; 3. Bos and Bison are united within the tribe Bovini by the presence of shared derivative submetacentric X chromosomes; 4. Sika and white-tailed deer X chromosomes differ by inversion from X chromosomes of the Bovinae; 5. The Bovini ancestral Y chromosome was probably a small acrocentric; 6. Bos taurus, B. gaurus and B. banteng share derivative metacentric Y chromosomes; 7. Syncerus and Bubalus are united by the acquisition of X-specific repetitive DNA sequence on their Y chromosomes; 8. Bovinae and Cervidae X chromosome centromere position varies without concomitant change in locus order. Preliminary data indicate that a knowledge of the chromosomal distribution of NORs among the Bovidae will prove to be phylogenetically informative.

Cytogenetic alignment of the bovine chromosome 13 genome map by fluorescence in-situ hybridization of human chromosome 10 and 20 comparative markers.

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of six genes (IL2RA, VIM, THBD, PLC-II, CSNK2A1 and TOP1) previously assigned to human chromosomes 10 or 20 (HSA10 or HSA20). Four of the genes were found represented in the bovine BAC library by at least one clone. The identified BAC clones were used as probes in single-color fluorescence in-situ hybridization (FISH) to determine the chromosomal band location of each gene. As predicted by the human/bovine comparative map and comparative chromosome painting analysis, the four genes mapped to bovine chromosome 13 (BTA13). Dual-color FISH was then used to integrate these four type I markers into the existing BTA13 genome map. These FISH results anchor the BTA13 genome map from bands 14-23, and confirm the presence of a conserved HSA10 homologous synteny group on BTA13 centromeric to a HSA20 homologous segment.

Physical assignment of microsatellite- containing BACs to bovine chromosomes.

Here we report the physical assignment of 40 microsatellite markers by fluorescence in situ hybridization to 13 different bovine chromosomes. This information will be valuable in providing physically anchored landmarks for the construction of contigs throughout the bovine genome. It also is useful for the purpose of integrating the linkage maps of these chromosomes to their physical maps and determining the physical coverage of these linkage groups.

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization.

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type 1 loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type 1 loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere-PRNP-(SOD1L/AVP/OXT)-(BL42/GNAS1)- HCK-CSSM30. The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.

Physical assignment of six type I anchor loci to bovine chromosome 19 by fluorescence in situ hybridization.

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library (NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere-NF1-CRYB1-CHRNB1-TP53-GH1-P4HB, was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53, previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14-22.

A karyotypic analysis of nilgai, Boselaphus tragocamelus (Artiodactyla: Bovidae).

A combination of chromosomal banding and fluorescence in situ hybridization (FISH) was used to characterize the karyotype of Boselaphus tragocamelus (nilgai) relative to the domestic cattle standard karyotype. G-, Q- and C-band karyotypes of nilgai are presented, and the chromosomal complement of nilgai is determined to be 2n=46 (female FN=60, male FN=59; NAA=56), consistent with previous reports for the species. Comparisons with cattle identified extensive monobrachial homologies with some noteworthy exceptions. Chromosome 25 is centrically fused to 24, and chromosome 16 is acrocentric. Both appear to have additional pericentromeric material not seen in the equivalent cattle acrocentrics. This pericentromeric chromatin may be the result of de novo additions or translocation of pericentromeric material from chromosome 6, which is shown to be centrically fused to 13 but is only about two-thirds the length of cattle 6. Comparisons with cattle demonstrated that nilgai chromosome 17 has undergone a paracentric inversion and that chromosome 20 has two blocks of interstitial constitutive heterochromatin. The identities of both chromosomes were confirmed by chromosomal FISH. Furthermore, chromosomal banding and FISH were used to determine that autosome 14 has been fused to the ancestral X and Y of nilgai to form compound neo-X and -Y chromosomes. Additional FISH analyses were conducted to confirm other proposed chromosome homologies and to identify nucleolar organizing regions within the nilgai complement.