A linkage group of five DNA markers on human chromosome 10

Five chromosome 10 DNA markers (D10S1, D10S3, D10S4, D10S5, and RBP3) were typed in five large pedigrees with multiple endocrine neoplasia type 2A (MEN-2A) and in five non-MEN-2A pedigrees. Linkage analyses showed that these loci and the locus for MEN-2A (MEN2A) are in one linkage group spanning at least 70 cM. The order of the marker loci is RBP3-D10S5-D10S3-D10S1-D10S4, with interlocus recombination frequencies of 7, 13-19, 19, and 19%, respectively, all on the same side of MEN2A. Analyses of sex-specific recombination frequencies indicated no significant differences between males and females for any of the map intervals studied. Previous localization of D10S5 and RBP3 to the proximal region of the long arm and the pericentric region, respectively, comparison of results with other studies, and our preliminary results with other chromosome 10 markers suggest that the D10S4 end of the map extends into the long arm. Our linkage map has been constructed using only two- and three-locus analyses. It will be possible to combine our results with those of other groups to construct a more detailed and accurate genetic map of chromosome 10.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Optimal alignments in linear space

Space, not time, is often the limiting factor when computingoptimal sequence alignments, and a number of recent papers inthe biology literature have proposed space-saving strategies.However, a 1975 computer science paper by Hirschberg presenteda method that is superior to the new proposals, both in theoryand in practice. The goal of this paper is to give Hirschberg'sidea the visibility it deserves by developing a linear-spaceversion of Gotoh's algorithm, which accommodates affine gappenalties. A portable C-software package implementing this algorithmis available on the BIONET free of charge. Received on October 14, 1987; accepted on December 19, 1987     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

Recruiting physicians to rural practice. Suggestions for success.

Medical school graduates from 1986 to 1988 and current residents in 12 family practice residency programs in the Northwest (N = 302) were surveyed to identify important factors in the recruitment process for their first postresidency placement. The study sought to compare the recruitment practices of rural communities and urban sites. Specific questions addressed in the study concerned sources of information about practice opportunities, stage in training when job search was initiated, factors related to unsuccessful site visits, and activities scheduled in the visit. Results indicated that referrals from faculty were the most valued source of information. Most job searches were initiated in the first 6 months of the third year in training. An unreceptive physician community and a reluctant spouse or partner were substantial problems for residents making site visits to rural communities. Rural sites tended to provide a broader mix of professional and personal activities during the visit.     

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.     

Vicilin genes ofPisum

Summary We describe four genomic clones of pea 7S storage protein gene, one of which corresponds to convicilin, and the others to vicilin. Hybridization studies exploiting these clones, and previously identified cDNA clones, have enabled us to define six different loci. Three of these loci have been mapped to positions on chromosome 7.     

In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits

A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose. The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide.