The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

A familial "balanced" 3;9 translocation with cryptic 8q insertion leading to deletion and duplication of 9p23 loci in siblings.

A child with phenotypic features of the 9p- syndrome, including metopic craniosynostosis, small ears, abdominal wall defect, and mental retardation, as well as hypopigmentation, was found to have a cytogenetically balanced 3;9 translocation, with breakpoints at 3p11 and 9p23, inherited from his phenotypically normal father. Molecular analysis showed heterozygous deletion of the TYRP (tyrosinase-related protein) locus, as well as loci D9S157, D9S274, D9S268, and D9S267, in the child but in neither parent. FISH analysis of the proband''s father indicated that loci deleted in his son, including TYRP, were present on neither the der(3) nor the der(9) translocation products but had been inserted into the long arm of chromosome 8. Therefore, the apparent deletion of these loci in the proband was the result of meiotic segregation of the father''s 3;9 translocation chromosomes together with his normal chromosome 8 (not bearing the insertion from 9p23). Neither the deletion of these 9p23 loci from the translocation chromosomes nor their insertion into 8q was detectable by standard chromosome banding techniques. The proband''s sister exhibited speech delay, mild facial dysmorphism, and renal malformation, and her karyotype was 46,XX. Molecular analysis showed that she had inherited normal chromosomes 3 and 9, as well as the chromosome 8 with the insertion of 9p23 material, from her father.(ABSTRACT TRUNCATED AT 250 WORDS)     

Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFN secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.     

Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots

SUMMARY The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin-inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens . An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.     

Mutation analysis of UBE3A in Angelman syndrome patients.

Angelman syndrome (AS) is caused by chromosome 15q11-q13 deletions of maternal origin, by paternal uniparental disomy (UPD) 15, by imprinting defects, and by mutations in the UBE3A gene. UBE3A encodes a ubiquitin-protein ligase and shows brain-specific imprinting. Here we describe UBE3A coding-region mutations detected by SSCP analysis in 13 AS individuals or families. Two identical de novo 5-bp duplications in exon 16 were found. Among the other 11 unique mutations, 8 were small deletions or insertions predicted to cause frameshifts, 1 was a mutation to a stop codon, 1 was a missense mutation, and 1 was predicted to cause insertion of an isoleucine in the hect domain of the UBE3A protein, which functions in E2 binding and ubiquitin transfer. Eight of the cases were familial, and five were sporadic. In two familial cases and one sporadic case, mosaicism for UBE3A mutations was detected: in the mother of three AS sons, in the maternal grandfather of two AS first cousins, and in the mother of an AS daughter. The frequencies with which we detected mutations were 5 (14%) of 35 in sporadic cases and 8 (80%) of 10 in familial cases.     

Mucin-1-related T cell infiltration in colorectal carcinoma

 Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes’ B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes’ stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3⁺ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited. Received: 2 April 1996 / Accepted: 28 May 1996     

Evolution and biogeography of the austral genus Phyllocladus (Podocarpaceae)

Aim To infer evolutionary relationships within the genus Phyllocladus and among its close relatives by phylogenetic analysis of DNA sequences. Interpret the inferred relationships in association with the fossil record to examine the origin and diversification of the genus. Location Australasia. Methods Phylogenetic analyses of rbcL, matK and internal transcribed spacer (ITS) sequences representing all of the extant species of Phyllocladus and a selection of outgroups from Podocarpaceae and Araucariaceae. Results The rbcL and matK sequences exhibit little variation within Phyllocladus, but ally its members to Podocarpaceae although its immediate sister remains unclear. The ITS sequences resolve all five species of Phyllocladus and two intraspecific ecotypes of P. alpinus. Main conclusions Phyllocladus forms a distinct lineage that diverged early in the evolutionary history of Podocarpaceae. The fossil record indicates that the genus was more widely distributed and morphologically diverse during the early Tertiary than at present. Although of Mesozoic origin, the level of sequence variation within Phyllocladus suggests that the extant species radiated during the late Tertiary c. 6.3 ± 0.9 Ma. New Zealand is the present centre of species diversity.