Frameshift and splice-junction mutations in the sterol 27-hydroxylase gene cause cerebrotendinous xanthomatosis in Jews or Moroccan origin.

The sterol 27-hydroxylase (EC 1.14.13.15) catalyzes steps in the oxidation of sterol intermediates that form bile acids. Mutations in this gene give rise to the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). CTX is characterized by tendon xanthomas, cataracts, a multitude of neurological manifestations, and premature atherosclerosis. A relatively high prevalence of the disease has been noted in Jews originating from Morocco. The major objectives of the present investigation were to determine the gene structure and characterize the common mutant alleles that cause CTX in Moroccan Jews. The gene contains nine exons and eight introns and encompasses at least 18.6 kb of DNA. The putative promoter region is rich in guanidine and cytosine residues and contains potential binding sites for the transcription factor Sp1 and the liver transcription factor, LF-B1. Blotting analysis revealed that the mutant alleles do not produce any detectable sterol 27-hydroxylase mRNA. No major gene rearrangements were found and single-strand conformational polymorphism followed by sequence analysis identified two underlying mutations: deletion of thymidine in exon 4 and a guanosine to adenosine substitution at the 3' splice acceptor site of intron 4 of the gene. The molecular characterization of CTX in Jews of Moroccan origin provides a definitive diagnosis of this treatable disease.

Production of beta-lactamase by non-streptomyces Actinomycetales.

Supernatants and whole cells from fermentation broths of Micromonospora, Nocardia, Oerskovia, and other genera of the Actinomycetales were examined for the presence of beta-lactamase activity by using the chromogenic cephalosporin 87/312. Nearly 60% of the 250 isolates examined produced detectable levels of beta-lactamase. All enzyme preparations were active over a range of pH values from 6.5 to 8.2, with maximum activity occurring between 7.0 and 7.8. The preparations varied in their stability at 60 degrees C. An examination of selected enzyme preparations revealed a similarity between substrate specificities of the non-Streptomyces Actinomycetales and gram-negative-bacterial beta-lactamases.

Hypercholesterolemia in five Israeli Christian-Arab kindreds is caused by the "Lebanese" allele at the low density lipoprotein receptor gene locus and by an additional independent major factor.

Segregation analyses were performed for plasma low density lipoprotein cholesterol (LDL-C), triglycerides (TG) and high density lipoprotein cholesterol (HDL-C) in five Christian Arab kindreds identified through probands with familial hypercholesterolemia. In this subset of the Christian Arab community, the results were consistent with major gene determination of LDL-C with allele frequency (q) of 0.042 (95% confidence interval 0.008-0.079) in addition to polygenic transmission (h2 = 0.34). The "Lebanese" allele was identified directly by polymerase chain reaction and HinfI restriction analysis. Analysis of this mutation permits direct diagnosis of familial hypercholesterolemia in most affected individuals although our results indicated the possible existence of an additional independent factor leading to elevated LDL-C levels. The segregation results for TG indicated the presence of a major effect, although the existence of a major gene could not be demonstrated. There was also no evidence of a major locus effect on HDL-C levels.

A 69-kDa RNA-binding protein from Xenopus oocytes recognizes a common motif in two vegetally localized maternal mRNAs.

Vg1 mRNA, a maternal message encoding a member of the transforming growth factor beta superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nucleotide sequence has been identified in Vg1 RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, 991-994]. To understand how cis- and trans-acting factors are involved in Vg1 mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vg1 mRNA. S100 extracts of late-stage oocytes contain a protein-binding activity that protects specific regions of labeled Vg1 mRNA from degradation by RNase T1. The use of different regions of Vg1 RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vg1 message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the down-stream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor beta 5 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.

RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.

Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3' UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of beta actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.

Expression of lipoprotein lipase mRNA in rat heart is localized mainly to mesenchymal cells as studied by in situ hybridization.

The expression of lipoprotein lipase mRNA (LPL mRNA) was studied in rat hearts by use of a sulfur-35-labeled antisense mRNA probe. Rats were studied under three conditions: fed, fasted, and injected with cholera toxin (an irreversible agonist of adenylate cyclase) and then fasted. The highest LPL activity was found in the hearts of cholera toxin-injected, fasted rats. After injection of cholera toxin, LPL mRNA levels were 3.5-fold higher than those from fed rats. Using in situ hybridization, we studied the site of expression of LPL mRNA under the same three experimental conditions. In sections of hearts from cholera toxin-injected, fasted rats, concentrations of autoradiographic grains, representing the site of LPL mRNA, were seen over interstitial elements, which comprise capillary and perivascular cells. A more diffuse and sparse reaction was seen over cardiac myocytes and was not always distinguishable from background. A similar but much less definitive localization was seen in sections of hearts from fasted rats. The present results indicate that in the rat heart, the main site of LPL synthesis and processing, especially after stimulation with an irreversible agonist of adenylate cyclase, is localized to interstitial elements rather than to adult cardiac myocytes.