Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Plague Meningitis—A Retrospective Analysis of Cases Reported in the United States, 1970-1979

Meningitis caused by Yersinia pestis developed in 6 (6%) of a total of 105 patients with plague reported to the Centers for Disease Control from 1970 to 1979. Five of the six cases occurred in children aged 10 to 15 years. All six patients received antibiotic therapy before meningitis developed, which appeared between the 9th and 14th days after the onset of acute illness in five of the six patients. There were no neurologic sequelae. The antigenic and biochemical profiles of the Y pestis strains isolated from cerebrospinal fluid in the meningitis cases did not differ from those of the Y pestis strains obtained from blood and bubo aspirates in the other 99 patients, and neither did in vitro studies suggest antibiotic resistance. While plague meningitis is an uncommon complication of acute plague infection, physicians in the western United States should be aware that it may develop as much as 14 days after antibiotic therapy for the acute plague infection has been initiated.     

The mouse Fc receptor for IgG (Ly-17) : molecular cloning and specificity

A cDNA clone encoding the mouse Ly-17⁺ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional FcR investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG_1/2b and rabbit IgG complexes.     

Effect of gibberellic acid on pod set in soybean

Field-grown soybean plants (Glycine max (L.) Merr. cv. Evans) were treated with gibberellic acid (GA₃; 10gl^–1) and/or (2-chloroethyl)-trimethylammonium chloride (CCC; 0.8gl^–1) in 1983 and 1984, and subsequent anthesis, pod set, seed size, seed number, and seed yield were determined at one node. The treatments were applied to five leaves in the center of each plant (typically leaves 7–11) and reproductive development at the node in the center of those leaves was monitored. Gibberellin A₃ applied Early (about 3d before anthesis of the first flower at the monitored node) had no effect on the number of flowers produced, but decreased the fraction of flowers that set pods in both experimental years (by 32% in 1983 and 76% in 1984). Seed size was slightly decreased by the GA₃ treatment in 1983 but not in 1984. The Middle GA₃ treatment (applied about 3 days after the Early treatment) slightly decreased the number of pods set; and Late treatments (9 days after) had no effect. None of the monitored parameters were affected by CCC.The Early experiments were repeated with two additional genotypes, Lincoln and T210. Genotype T210 is a single-gene, dwarf mutant of Lincoln whose stem elongation and leaf expansion are insensitive to GA₃. Gibberellin A₃ affected the reproductive parameters in Lincoln very similarly to Evans but those in T210 were unaffected. This indicates that GA₃ exerts its effect by increasing the mass of vegetative tissue and thus diverting assimilates away from the pods. However, since the mutation in T210 might affect a receptor that is in flowers as well as shoots, it is possible that GA₃ exerted its effect on the normal genotypes directly on the developing pods, rather than indirectly by diverting photoassimilates.     

Peripheral oestrogen metabolism in postmenopausal women with or without breast cancer: the role of dietary lipids and growth factors

Studies we have carried out have revealed significant differences in oestrogen production and metabolism between normal women and postmenopausal women with breast cancer. The free, biologically available fraction of oestradiol is elevated in plasma from women with breast cancer and we have found that metabolic clearance rates and production rates of oestradiol are also increased. In vitro studies have suggested that lipids can influence the distribution of sex steroids in plasma and we have therefore examined the effect of dietary lipids on the distribution of sex steroids in plasma in vivo. Consumption of a meal with a high saturated fat content or the oral or i.v. administration of "Intralipid", a stabilised emulsion of soya bean oil that is high in unsaturated free fatty acids, had little effect on the available fractions of oestradiol in plasma. However, results from a preliminary study suggest that long-term changes in dietary fat intake can alter the distribution of steroids in plasma. It is concluded that dietary lipids may influence the availability of sex steroids to tissues. Such a mechanism could account for the significant correlation that has been found between dietary fat consumption and the incidence of breast cancer on a world-wide basis.     

DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.     

Transductional analysis of chromosome replication time

Summary Following transduction of exponentially growing cultures of Escherichia coli with phage P1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time. We show that transductants for markers located at different positions on the chromosome begin to increse at different times, in reverse order to that in which they are replicated. The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relationship to calculate the C-period of E. coli K12 growing at 30°C. We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited.     

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.