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Biomedical subjects

M H Meisler

Publications and source records attributed to M H Meisler.

11 recordsLinked to original sources

The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a.

Exon shuffling is thought to be an important mechanism for evolution of new genes. Here we show that the mouse neurological mutation flailer (flr) expresses a novel gene that combines the promoter and first two exons of guanine nucleotide binding protein beta 5 (Gnb5) with the C-terminal exons of the closely linked Myosin 5A (MyoVA) gene (Myo5a). The flailer protein, which is expressed predominantly in brain, contains the N-terminal 83 amino acids of Gnb5 fused in-frame with the C-terminal 711 amino acids of MyoVA, including the globular tail domain that binds organelles for intracellular transport. Biochemical and genetic studies indicate that the flailer protein competes with wild-type MyoVA in vivo, preventing the localization of smooth endoplasmic reticulum vesicles in the dendritic spines of cerebellar Purkinje cells. The flailer protein thus has a dominant-negative mechanism of action with a recessive mode of inheritance due to the dependence of competitive binding on the ratio between mutant and wild-type proteins. The chromosomal arrangement of Myo5a upstream of Gnb5 is consistent with non-homologous recombination as the mutational mechanism. To our knowledge, flailer is the first example of a mammalian mutation caused by germ line exon shuffling between unrelated genes.

Amino Acid Sequence

Localization of insulin-2 (Ins-2) and the obesity mutant tubby (tub) to distinct regions of mouse chromosome 7.

A DNA mapping panel derived from an interspecific backcross was used to position the mouse insulin-2 locus (Ins-2) on Chromosome 7, near H19 (0/114 recombinants) and Th (1/114 recombinants). Ins-2 is part of a human-mouse conserved linkage group that includes Th, H19, and Igf-2. Analysis of segregation in the F2 generation from the cross C57BL/6J-tub/tub x CAST/Ei demonstrated that Ins-2 and the obesity mutant tubby (tub) are distinct loci, thus eliminating Ins-2 as a candidate gene for tub. These results also refine the estimated genetic distance between tub and Hbb to 2.4 +/- 1.4 cM. The predicted location for a human homolog of tubby is HSA 11p15.

Animals

Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene.

The human salivary amylase genes are associated with two inserted elements, a gamma-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.

Actins

Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment.

Expression of the mouse pancreatic amylase gene Amy-2.2 is increased approximately 10-fold in response to increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequence mediating this response has been localized to the 5' flanking region of the amylase gene by analysis of hybrid constructs in transgenic mice. The results define a 127-base pair dietary response unit that includes two previously described regulatory elements, an insulin-responsive element and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences. Another mouse amylase gene Amy-2.1 is only minimally responsive to insulin and to diet. The data are consistent with the hypothesis that the insulin-response element is necessary but not sufficient for regulation of amylase by dietary carbohydrate.

Amylases

A sensitive fluorescent assay for N-acetyltransferase activity in human lymphocytes from newborns and adults.

We have developed a simplified assay for the enzyme N-acetyltransferase, based upon the loss of fluorescence after acetylation of the substrate p-aminobenzoic acid. This method is sufficiently sensitive to permit the quantitation of N-acetyltransferase activity in 10(5) human lymphocytes. Using this method, we have compared the level of N-acetyltransferase activity in lymphocytes from adult peripheral blood and from cord blood samples.

Acetyltransferases

GM1-gangliosidosis: chromosome 3 assignment of the beta-galactosidase-A gene (beta GALA).

The structural gene (beta GALA) coding for lysosomal beta-galactosidase-A (EC 3.2.1.23) has been assigned to human chromosome 3 using man--mouse somatic cell hybrids. Human beta-galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver beta-galactosidase-A. The antiserum precipitates beta-galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with GM1 gangliosidosis. We have analyzed 90 primary man--mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for beta GALA assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of beta GALA to chromosome 3; all other chromosomes were excluded. The evidence suggests that GM1 gangliosidosis is a consequence of mutation at this beta GALA locus on chromosome 3.

Animals

Synthesis and secretion of kidney beta-galactosidase in mutant le/le mice.

We have found that the pigmentation mutant light ear in the mouse has a striking effect on lysosomes in the kidney. Male mice homozygous for the le mutant allele have a 4-fold elevated concentration of kidney beta-galactosidase (EC 3.2.1.23). The abnormal elevation of kidney beta-galactosidase is the net result of two processes. First, beta-galactosidase is elevated due to the defective urinary secretion of lysosomal enzymes which is a specific effect of the le mutation. Secondly, this effect is most evident in males and testosterone-treated females because of the induction of beta-galactosidase synthesis by testosterone, which occurs in +/+ as well as in mutant mice. The pale-ear mutation (ep) which mimics the pigmentation phenotype of le also has a similar effect on kidney lysosomes.

Aging

Effects of the Bgs locus on mouse beta-galactosidase.

The Bgs locus determines tissue levels of beta-galactosidase in the mouse, so that enzyme levels are twice as high in mice carrying the Bgsh allele as in mice carrying the Bgsd allele (Felton et al., 1974). By immunotitration with antiserum to purified beta-galactosidase, we have found that the Bgs locus influences the amount of enzyme protein present in the tissues. We have utilized recombinant inbred lines derived from a cross between C57BL/6J and DBA/2J mice to confirm the location of the Bgs locus on chromosome 9. The inhibition of mouse beta-galactosidase by the active-site-directed reagent N-bromoacetyl-beta-D-galactosylamine has been investigated. beta-Galactosidase from the high and low Bgs strains has identical affinity for this inhibitor.

Acetylgalactosamine

Inhibition of human liver beta-galactosidases and beta-glucosidase by n-bromoacetyl-beta-D-galactosylamine.

N-Bromoacetyl-beta-D-galactosylamine is an irreversible inhibitor of the 'acid' and the 'neutral' beta-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) of human liver. The inactivation of acid beta-galactosidase appears to involve a group with a pKa = 4.5. The inhibition of neutral beta-galactosidase only occurs above pH 8.0. Both enzymes are protected against inhibition by the presence of substrates, suggesting that the inhibitor reacts with the active site of the enzymes. Other lysosomal hydrolases are not inhibited by N-bromoacetyl-beta-D-galactosylamine, with the exception of 'neutral' beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21). The pH dependence of neutral beta-glucosidase inactivation is essentially identical to that of the neutral beta-galactosidase. Inhibition of beta-glucosidase by this galactose derivative suggests that the same enzyme may bind glucosides and galactosides. Furthermore, both neutral beta-galactosidase and beta-glucosidase are inactivated at 52 degrees C with a half-life of 7.5 min. The presence of a single enzyme with both beta-glucosidase and beta-galactosidase activities is also supported by mixed-substrate experiments.

Acetylgalactosamine