Single nucleotide polymorphisms in cytochrome P450 genes from barley.

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.

Cell Suspensions from Collagenase Digestion of Bone Marrow Trephine Biopsy Specimens-its Use in Lymphoma.

Collagenase digestion allows cells to be released into suspension from bone marrow tissue. Discrete abnormal populations of lymphoid cells can be identified by cell morphology and immunological phenotyping techniques. Viable cells are also available for chromosomal analysis. This technique makes cells available for analysis in cases of dry bone marrow taps and has a particular use in the investigation of bone marrow involvement by malignant lymphoma.

Cell suspensions from collagenase digestion of bone marrow trephine biopsy specimens.

A technique for the extraction of cells from bone marrow trephine core biopsy specimens using collagenase digestion was assessed in 39 cases (33 diagnostic and six normal). Diagnostically useful numbers of cells were extracted from all marrows. Morphological assessment of cytocentrifuge preparations of these cells gave a correct diagnosis in 23 (60%) of cases compared with 27 (70%) for the corresponding aspirated marrow smears. Phenotypic analysis using flow cytometry showed persistence of a range of surface membrane antigens following collagenase digestion. Increased autofluorescence was a problem in some cases. Cytochemistry, bone marrow culture, and cytogenetic analysis could also be carried out on these cells. It is concluded that this technique has useful diagnostic applications in cases of dry taps.

Enhanced expression of insulin-like growth factor II is not a necessary event in Wilms' tumour progression.

Wilms' tumour (WT) is a paediatric kidney tumour arising from the embryonal metanephrogenic blastema. Recent reports suggest that the expression of messenger RNA (mRNA) for insulin-like growth factor II (IGFII) is elevated in WT. Total cytoplasmic RNA was extracted from 11 sporadic WTs and analysed for IGFII mRNA using dot-blot hybridization. The level of IGFII mRNA expression varied greatly and not all tumours displayed enhanced IGFII expression. Two successive WT xenografts were established in nude mice. The original WT and first passage xenograft showed a blastematous histology, while the second passage xenograft showed epithelioid differentiation and tubule formation. Analysis of the expression patterns of these xenografts showed elevated IGFII expression in the primary undifferentiated tumour and the second differentiated xenograft, while the first undifferentiated xenograft failed to exhibit enhanced IGFII expression. These data show that elevated IGFII mRNA is not an essential component of the progression of WT and that WT tumourigenicity is independent of the level of IGFII expression. Therefore, IGFII overexpression in WT is most likely a tumour epiphenomenon.

Histopathological and tissue culture studies of a melanizing cell line derived from a retinoblastoma.

Histological examination of the enucleated eye of a 7-mth-old child revealed a retinoblastoma with areas of rosette formation as well as focal areas of melanin pigmentation. Biopsy derived cells readily established a continuous cell line in liquid culture. The cells which have now been cultured continuously for over 3 yr, were shown to be malignant by being non-contact inhibitable, by readily forming colonies in semi-solid nutrient agar medium and by producing tumours in nude mice. When grown to the point of overcrowding in liquid culture the cells became heavily melanized. Electron microscopy of the melanized cultured cells showed the melanin to be contained in melanosomes. These findings suggest that retinoblastomas may be derived from bipotential primitive retinal cells which retain the capacity for both nuclear cell and pigment cell differentiation. Alternatively, separate malignant transformations may have occurred in each of 2 different progenitor cell types committed to a separate differentiation pathway. The clinical behaviour of this tumour has not differed from that expected of non-pigmented retinoblastomas.

The effect of dietary restriction, adrenaline, hydrocortisone and surgery on the rates of death of 125IUdR-labelled, intravenously injected tumour cells in the lungs of mice.

Dietary restriction, adrenaline hydrocortisone or surgery reduced the rate at which pulmonarily arrested 125IUdR-labelled murine tumour cells were lost within 7 h of intravenous (i.v.) injection. Mice that had been adrenalectomised 10 days previously showed a normal intrapulmonary tumour cell loss rate with further surgery reducing this rate to approximately half that observed in normal mice that had been subjected to surgery. Thus, although it is likely that adrenal hormones play an important role in decreasing the rate of early intrapulmonary tumour cell loss, additional factors must be implicated. Mice subject to dietary restriction, adrenaline, hydrocortisone or surgery had reduced levels of in vitro growth inhibitor(s) in their sera. Despite this, individual surgically treated animals showed no correlation between serum in vitro-growth inhibitor levels and rate of loss of i.v. injected tumour cells from the lungs. Furthermore, the 24 h pre-incubation of tumour cells in inhibitor-rich serum did not influence the subsequent loss rate of such cells following i.v. injection into mice. Electron microscopic studies indicated that dietary restriction, adrenaline and surgery reduced the rate of intravascular tumour cell death. The decreased tumour cell death rate in mice receiving these treatments could not be related, however, to any consistent morphological change in the pulmonary vasculature. The decreased rate of intravascular tumour cell death in treated mice was followed by an increased number of lung tumours with only one of the tumour lines studied, indicating that the intravascular death rate need not be a major determinant of pulmonary tumour incidence.

Inhibition of the growth of murine tumour cells in vitro by serum from non-immune syngeneic and allogeneic mice.

Sera from DBA/2 and Quackenbush mice (which are non-immune for mastocytoma and Sarcoma 180 respectively) contain a heat-labile (56 degrees for 30 min) component(s) that inhibits the in vitro growth of DBA Mastocytoma P-815 X-2 and Sarcoma 180. Adsorption of the sera with tumour cells at 4 degrees did not eliminate the factor(s), suggesting that it is not an antibody. In liquid suspension cultures inhibitory activity was observed at concentrations of mouse serum of 10--20% and in semisolid agar clonogenic cell assays at concentrations as low as 1%. The influences of the inhibitor(s) for both tumours and in both culture systems were parallel. However, there was a quantitative difference in susceptibility to other environmental factors (FCS concentration, bicarbonate concentration, and O2 tension) between the two tumours. These results parallel the in vivo findings where intravenously injected mastocytoma cells produced more tumours than did Sarcoma 180.