Frances Benham

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11g13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MENI-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM-SEA-D11S913-D115970-D11S97-D11S146-INT2-D11S971-D11S533-11gter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding μ-calpain (CAPN1) located CAPN1 to llg13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11g13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.

Heterogeneity of Alkaline Phosphatases in Different HeLa Lines

Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability,l-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALPstructural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the “derepressed” placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.

Three DNA markers for hypophosphataemic rickets

SummaryThis paper presents three markers, 16D/E, pHMAI (DXS208), and CRI-L1391 (DXS274), that show close linkage for X-linked hypophosphataemic rickets (HYP). DXS274 is closely linked to HYP (θmax= 0.00, Zmax = 4.20), and DXS41 (99.6), (θmax= 0.00, Zmax = 5.20). Marker 16D/E maps distal to the disease locus (θmax= 0.05, Zmax = 3.11). The pHMAI probe recognises the same restriction fragment length polymorphism (RFLP) as 99.6. Multipoint analysis suggests that the most probable order of loci is Xpter-(DXS43, 16D/E)-HYP-DXS274-(DXS208, DXS41)-Xcen. The location of DXS274 distal to HYP cannot be excluded, as no recombinants were observed between DXS274 and HYP, or between DXS274 and DXS41/DXS208. One of the families contains a large number of recombinants, four of which are double recombinants. This most probably means that the disease in this family maps elsewhere on the X chromosome or on an autosome, indicating locus heterogeneity.

Use of Alu-PCR to characterize hybrids containing multiple fragments and to generate new Xp21.3–p22.2 markers

Irradiation fragment hybrids potentially provide highly enriched sources of region-specific human DNA. However, such hybrids often contain multiple human pieces, not all of which can be easily detected. To develop specific resources for rapidly generating markers from Xp21.3-p22.2, we have single cell cloned two previously constructed irradiation hybrids that contain markers in this region and have achieved segregation of the different known fragments originally retained. Alu-PCR products were generated from subclones positive or negative for Xp21.3-p22.2 markers, and comparison of the ethidium bromide patterns between sister subclones facilitated identification of bands likely to map to particular regions; in contrast, subclones that shared markers but were derived from independent lines showed no overlap in ethidium bromide pattern. All Alu-PCR products from one subclone, 50K-19E, in which only three closely linked markers were detected (DXS41, DXS208, DXS274) were mapped back to their region of origin. Of 28 products, 15 mapped to Xp21.2-p22.2, and these make up a new set of regionally assigned markers. However, the mapping data identified four separate Xp fragments in 50K-19E, only one of which had been picked up by marker analysis. Mapping back gel-isolated Alu-PCR products from an irradiation hybrid prior to any cloning or screening generates a comprehensive profile of the human DNA retained and permits rapid selection of sequences derived only from the region of interest.

Alkaline phosphatase phenotypes in tumour and non-tumour cell lines: not an invariable marker for neoplastic transformation.

The cytochemical localisation and presumed isoenzyme type (based on selective inhibition experiments) of alkaline phosphatase in 5 cell lines derived frrom normal human, rat, mouse and hamster tissues, 6 human lymphoblastoid lines and 6 human and mouse tumour-derived cell lines are described. Enzyme activity varied between the cell lines. An isoenzyme inhibited by L-phenylalanine was present in 3 normal lines, 3 lymphoblastoid lines and 2 tumour lines. The presence of this isoenzyme cannot be used as a marker of neoplastic transformation.

Cosmid contigs spanning 9q34 including the candidate region for TSC1.

The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34.

Alkaline phosphatase in mitochondria

In electron microscope cytochemical studies alkaline phosphatase activity was present in the mitochondria of all liver cells and associated with the plasma membrane of the cells of bile canaliculi. The mitochondrial activity was partially inhibited by L-phenylalanine and Levamisole but the plasma membrane associated activity was completely inhibited by Levamisole. Biochemical assays have shown that a significant amount of the total mouse liver alkaline phosphatase activity was present in the mitochondria fraction. Starch gel electrophoresis showed that this mitochondrial alkaline phosphatase had a characteristic isoenzyme pattern, consisting of 3 distinct bands which were not retarded by neuraminidase treatment. The enzyme in the mitochondria-free supernatant showed one wide band which was retarded by neuraminidase.