Mathematical modeling accurately predicts the dynamics and scaling of nuclear growth in discrete cytoplasmic volumes.

Scaling of nuclear size with cell size has been observed in many species and cell types. In this work we formulate a modeling framework based on the limiting component hypothesis. We derive a family of spatio-temporal mathematical models for nuclear size determination based on different transport and growth mechanisms. We analyse model properties and use in vitro experimental data to identify the most probable mechanism. This suggests that nuclear volume scales with cell volume and that a nucleus controls its import rate as it grows. We further test the model by comparing to data of early frog development, where rapid cell divisions set the relevant time scales.

A deletion unit on chromosome 17q in epithelial ovarian tumors distal to the familial breast/ovarian cancer locus.

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have suggested that chromosome 17q may be the location of a gene of importance in ovarian carcinogenesis. We have examined tumor and normal DNA samples from 120 patients with ovarian tumors for allelic deletion at 12 loci on chromosome 17q. Allelic deletion was observed in 64 cases (53%) of which 56 showed loss of heterozygosity at all loci analyzed on 17q. The pattern of allele loss at metastatic sites was consistent with loss of heterozygosity having occurred prior to metastasis. A common region of deletion, defined by 6 cases of invasive epithelial ovarian cancer and a benign serous cystadenoma, spanned 16 cM and was delimited by nm23 and GH. This region is distal to the region on chromosome 17q to which the familial breast/ovarian cancer susceptibility gene has been mapped. The results suggest that a tumor suppressor gene involved in sporadic ovarian carcinogenesis is located on the distal portion of chromosome 17q and is distinct from the gene linked to familial cases.

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427, clone 221a. This cloned stock is most commonly used in research laboratories in genetic manipulation experiments and in studies of antigenic variation. Using 116 previously characterised chromosome-specific markers, we identify 11 diploid pairs of megabase chromosomes and detect no loss of synteny in EST and gene marker distribution between this stock and the genome project reference stock TREU 927/4. Nevertheless, the chromosomes of 427 are all larger than their homologues in 927, except chromosomes IIa and IXa. The greatest size variation is seen in chromosome I, the smallest of which is 1.1 Mb (927-Ia) and the largest 3.6 Mb (427-Ib). The total nuclear DNA content of both stocks has been estimated by comparison of the mobility of T. brucei and yeast chromosomes. Trypanosomes of stock 427 contain approximately 16.5 Mb more megabase chromosomal DNA than those of stock 927. We have detected the presence of bloodstream-form expression-site-associated sequences on eight or more megabase chromosomes. These sequences are not found on the same chromosomes in each stock. We have determined the chromosomal band location of nine characterised variant surface glycoprotein genes, including the currently expressed VSG 221. Our results demonstrate both the stability of the T. brucei genome, as illustrated by the conservation of syntenic groups of genes in the two stocks, and the polymorphic nature of the genomic regions involved in antigenic variation. We propose that the chromosomes of stock 427 be numbered to correspond to their homologues in the genome project reference stock TREU 927/4.

Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase of Trypanosoma brucei, GRESAG4.4B.

In this study, we describe the isolation and characterization of a new adenylyl cyclase from Trypanosoma brucei and its activation by dimerization of the catalytic domain. In agreement with the current nomenclature of trypanosomal adenylyl cyclases, this new gene is termed GRESAG4.4B. The complete ORF of the GRESAG4.4B gene encodes a protein of 1291 amino acids. Its predicted protein structure is consistent with the structure of other trypanosomal cyclases, and with the cyclases of L. donovani. GRESAG 4.4B is constitutively expressed during the life cycle of trypanosomes. GRESAG4.4B is a member of a gene family, which contains at least six members, which are all clustered on chromosome IV. The catalytic domain of GRESAG4.4B is able to dimerize spontaneously. However, these spontaneously formed, stable dimers only show minimal enzymatic activity. The addition of a leucine zipper (LZ) derived from the S. cerevisiae GCN 4 gene to the N-terminus of the catalytic domain of GRESAG4.4B strongly activated its enzymatic activity. The LZ appears to enforce a distinct conformation of the dimer, which leads to an increased enzymatic activity, and thus may mimic the effect of ligand-induced dimerization of adenylyl cyclase in vivo.

Analysis of ploidy (in megabase chromosomes) in Trypanosoma brucei after genetic exchange.

The megabase chromosomes of Trypanosoma brucei are normally diploid, but the extent to which this ploidy is maintained when parasites undergo genetic exchange is not known. To investigate this issue, a panel of 30 recombinant clones resulting from the co-transmission through tsetse flies of three different parental T. brucei lines in all pair-wise combinations (STIB 247, STIB 386 and TREU 927/4) were examined. These clones are products of 28 different mating events; four of them result from self-fertilisation and the others are F1 hybrids. DNA contents of the three parental lines were determined by flow cytometry and shown to differ only slightly with DNA content increasing in the order 927/4 < 247 < 386. Flow cytometry of the recombinant clones indicated DNA contents were similar to the parents in 28 clones and raised approximately 1.5 times the parental values in only two. The two F1 hybrid progeny with raised DNA contents were shown by marker analysis to be trisomic for seven independent loci indicating that they were probably triploid whereas progeny with DNA contents similar to parental values inherited a single allele from each parent for four independent loci indicating that they were diploid.

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock TREU927/4 (927). We have identified 11 diploid chromosomes ranging in size from 1 to 5.2 Mb approximately and pairs of homologues differ in size by up to 15%. A total of 401 cDNA probes were hybridised to T. brucei stock 927 chromosomes and 168 chromosome-specific markers were defined. Most of these markers were hybridised to the separated chromosomal DNA of two other cloned field isolates and four F1 progeny clones from a laboratory cross. The chromosomes vary in size by up to two and a half times between stocks and the DNA content of the 11 pairs of homologues varies by up to 33% in different stocks. Stock 927 contains the smallest chromosomes and the least nuclear genomic DNA. Nevertheless, all 11 syntenic groups of cDNA probes are maintained in all stocks. In the F1 hybrids only we have identified one extra PFG band to which none of our probes hybridise. We have shown that probes thought to be specific for the bloodstream-form variant surface glycoprotein expression sites hybridise to different chromosomes in different stocks and may hybridise to either one or both of a homologous pair of chromosomes. We have also determined the chromosomal location of the ribosomal RNA gene arrays.

T cell receptor gamma/delta expression on lymphocyte populations of breast cancer patients.

The quantitative distribution and phenotype of gamma/delta lymphocytes in the peripheral blood (PBL), tumour draining lymph node (LNL) and tumour infiltrating lymphocytes (TIL) from breast carcinoma patients were determined by one- and two-colour flow cytometry. The TCR-gamma/delta + cells generally expressed the T cell lineage antigen CD3. The proportions of such cells were variable but generally small from all the three sources. Phenotypic analysis revealed that the CD8 marker was consistently and predominantly observed on gamma/delta + CD3+ cells in the tumour infiltrate, whereas CD4 expression, while generally low, was noted on a significant percentage (median 10%) of LNL gamma/delta + lymphocytes. In both PBL and LNL the predominant gamma/delta cell population was CD4-8-.

Polymerase chain reaction allelotyping of human ovarian cancer.

We have used a set of microsatellite polymorphisms (MSPs) to examine the location and frequency of allele loss throughout the genome in a panel of 25 human epithelial ovarian tumours. When more than one MSP was employed per arm, mean informativity was 85.2% (range 64-100%). The average fractional allelic loss was 0.28 (range 0-0.65). A high frequency of allele loss was seen at 5q (40%), 9q (48%), 11p (43%), 14q (46%), 15q (40%), 17p (61%), 17q (64%), 19p (45%) and Xp (40%), confirming previous findings at some sites, but also suggesting the existence of new tumour-suppressor genes in regions (9q, 14q, 15q) which have not previously been studied in ovarian cancer. For 9q and 14q, partial loss of the arm was more common than loss of heterozygosity for all loci. There was a significant relationship between allele loss affecting the short arm of chromosome 17 and allele loss affecting 17q (P < 0.001). No other relationship was detected between allele losses at different sites. Polymerase chain reaction allelotyping is suitable for the examination of very small tumour samples and tumours in which classical karyotyping is problematic.