T-cell receptor gene rearrangement in primary tumors: effect of genetic background and inducing agent

The status of T-cell receptor beta and gamma genes has been assessed in a series of primary tumors induced by a chemical carcinogen or by gamma-irradiation using two inbred strains of mice. It appears that these well-characterized regimens of carcinogenesis yield T-cell tumors showing gene rearrangements consistent with a clonal origin of the tumors. Individual rearranged bands seem to represent orthodox, intralocus recombination events. A variety of rearrangement phenotypes are observed, most strikingly for the gamma genes, and differences in the degree of T-cell receptor gene rearrangements observed can be categorized according to the inducing agent and to the genetic background of the mice, with the implication that premalignant thymocytes have been captured in different stages of T-cell development. Additionally, primary tumors were shown to express significant levels of mature beta gene mRNA.     

Variation in human mate choice: simultaneously investigating heritability, parental influence, sexual imprinting, and assortative mating

Human mate choice is central to individuals' lives and to the evolution of the species, but the basis of variation in mate choice is not well understood. Here we looked at a large community-based sample of twins and their partners and parents ([Formula: see text] individuals) to test for genetic and family environmental influences on mate choice, while controlling for and not controlling for the effects of assortative mating. Key traits were analyzed, including height, body mass index, age, education, income, personality, social attitudes, and religiosity. This revealed near-zero genetic influences on male and female mate choice over all traits and no significant genetic influences on mate choice for any specific trait. A significant family environmental influence was found for the age and income of females' mate choices, possibly reflecting parental influence over mating decisions. We also tested for evidence of sexual imprinting, where individuals acquire mate-choice criteria during development by using their opposite-sex parent as the template of a desirable mate; there was no such effect for any trait. The main discernible pattern of mate choice was assortative mating; we found that partner similarity was due to initial choice rather than convergence and also at least in part to phenotypic matching.     

Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.     

Micro RNA, C/D RNA and parental genoming imprinting.

Experimental and computer-assisted approaches have led us to identify hundreds of small non-coding RNA (ncRNAs) whose genes - clustered at two chromosomal domains (the DLK1-GTl2 region/human 15q11q13 and the SNURF-SNRPN/human 14q32 loc) - are subjected to genomic imprinting. Here, we discuss their genomic organization, their expression pattern and their potential functions. A travers des approches in silico et expérimentales, nous avons récemment identifié des centaines de petits ARN non-codants, ARNnc - ARN C/D et microARN - dont les gènes sont soumis à l'empreinte génomique parentale (EGP). Ils sont regroupés au sein de deux loci chromosomiques conservés: le domaine DLK1-GTL2 (locus Callypige, position chromosomique humaine 14q32) et le domaine SNURF-SNRPN (locus Prader-Willi, position chromosomique humaine 15q11q13). Dans cet article, l'organisation génique, le mode d'expression et les fonctions de ces petits ARN sont présentés.     

Equal parental origin of chromosome 22 losses in human sporadic meningioma: no evidence for genomic imprinting.

Inactivation of tumor suppressor genes can occur either by mutation at the gene locus or by loss of part or all of the chromosome region containing the gene. The latter is most frequently detected by DNA markers as loss of heterozygosity in the tumor tissue. In several reports, the paternal homologue was preferentially retained in embryonal tumors associated with loss of particular chromosomal regions, suggesting genomic imprinting of the corresponding tumor suppressor loci. To explore the generality of these findings and the possible role of genomic imprinting in adult tumors of the nervous system, we have determined the parental origin of chromosome 22 loss in sporadic meningioma. Of nine cases studied, five tumors retained the maternally derived chromosome 22 homologue while four retained the paternally derived chromosome 22. Thus, in contrast to the embryonal tumors, the meningioma locus on chromosome 22 is inactivated by random mutation in sporadic adult meningiomas.     

Cloning of an activated human ret gene with a novel 5' sequence fused by DNA rearrangement.

By transfecting a high-Mr DNA from human stomach cancer into NIH3T3 cells, a transforming sequence that showed homology with the human ret gene was identified. The transforming sequence was found to be generated by a DNA rearrangement in the human ret proto-oncogene. This rearrangement was suggested to have occurred during the transfection procedure. The nucleotide sequences of cDNAs of the rearranged ret gene and deduced amino acid (aa) sequences revealed that the rearrangement had resulted in recombination of the 3' segment of the ret proto-oncogene with a segment of an unknown human sequence, and that the recombination had generated a novel gene encoding a fusion protein of 435 aa. The rearrangement was presumed to be responsible for activation of the ret gene.     

Alterations of expression level of RASSFIA gene in primary epithelial tumors of various locations

Tumor-suppressor activity was established for RASSF1A gene by in vitro and in vivo including studies of knock-out mutated mice cells. Data on methylation of promoter region and expression decrease revealed mainly in cancer cell lines were reported. Here, analysis of RASSF1A mRNA quantity was performed for the first time in primary epithelial malignant tumors of five various locations from 130 patients by semi-quantitative RT-PCR. Representative sets of kidney, lung and breast carcinomas samples were studied. Preliminary data for RASSF1A expression in ovarian and colorectal carcinomas are also reported. Our system studies showed unexpected expression profiles, namely mRNA level increase more frequently (2-7 times) than decrease in renal, breast, ovarian, and colorectal carcinomas. Increasing RASSF1A mRNA level was revealed significantly more frequently in renal cell carcinoma (24/38, 63% vs. 8/38, 21%, P = 0.0004, by Fisher exact test) and ovarian carcinomas (8/13, 62% vs. 2/13, 15%, P = 0.0114). Only in non-small cell lung cancer decreasing and increasing of RASSF1A expression were observed with equal frequency (16/38, 42%). Noteworthy, for early clinical stages prevalence of increasing expression both in squamous cell lung cancer and in adenocarcinoma was revealed, and for advanced clinical stages evident prevalence of decreasing RASSF1A expression was established. Cases with increasing expression both in early and advanced stages of clear cell renal cell carcinoma were in prevalence, in advanced stages it was proved significantly (P = 0.0094). These data suggested that RASSF1A expression alterations were tumor specific. Mentioned above regularity could point onto ambivalent RASSF1A functions in tumors--a tumor-suppressor gene and a proto-oncogene as well.     

Genetic determinants of feline leukemia virus-induced lymphoid tumors: patterns of proviral insertion and gene rearrangement.

The genetic basis of feline leukemia virus (FeLV)-induced lymphoma was investigated in a series of 63 lymphoid tumors and tumor cell lines of presumptive T-cell origin. These were examined for virus-induced rearrangements of the c-myc, flvi-2 (bmi-1), fit-1, and pim-1 loci, for T-cell receptor (TCR) gene rearrangements, and for the presence of env recombinant FeLV (FeLV-B). The myc locus was most frequently affected in naturally occurring lymphomas (32%; n = 38) either by transduction (21%) or by proviral insertion (11%). Proviral insertions were also common at flvi-2 (24%). The two other loci were occupied in a smaller number of the naturally occurring tumors (fit-1, 8%; pim-1, 5%). Examination of the entire set of tumors showed that significant numbers were affected at two (19%) or three (5%) of the loci. Occupation of the fit-1 locus was observed most frequently in tumors induced by FeLV-myc strains, while flvi-2 insertions occurred with similar frequency in the presence or absence of obvious c-myc activation. These results suggest a hierarchy of mutational events in the genesis of feline T-cell lymphomas by FeLV and implicate insertion at fit-1 as a late progression step. The strongest links observed were with T-cell development, as monitored by rearrangement status of the TCR beta-chain gene, which was positively associated with activation of myc (P < 0.001), and with proviral insertion at flvi-2 (P = 0.02). This analysis also revealed a genetically distinct subset of thymic lymphomas with unrearranged TCR beta-chain genes in which the known target loci were involved very infrequently. The presence of env recombinant FeLV (FeLV-B) showed a negative correlation with proviral insertion at fit-1, possibly due to the rapid onset of these tumors. These results shed further light on the multistep process of FeLV leukemogenesis and the relationships between lymphoid cell maturation and susceptibility to FeLV transformation.     

HER-2/neu gene in primary and local metastatic axillary lymph nodes in human breast tumors.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.     

Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum.

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.     

Oncogene alterations in primary,recurrent, and metastatic human bone tumors

We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors—osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients)—in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease. © 1996 Wiley-Liss, Inc.     

Mutations of the P53 gene, including an intronic point mutation, in colorectal tumors.

In this study, analysis of structural changes of the p53 gene in colorectal tumors revealed point mutations detected in 8 of 14 carcinomas and 2 of 2 adenomas. Of these 10 cases with point mutations, eight had one or more missense mutations, one had a nonsense mutation, and the remaining one had, interestingly, an intronic point mutation with subsequent activation of a cryptic splice donor site in the flanking exon. This report contains the first identification of an intronic point mutation of the p53 gene in a colorectal cancer case.     

Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.