Impaired prenylation of Rab GTPases in the gunmetal mouse causes defects in bone cell function.

Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function.

Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo.

Nitrogen-containing bisphosphonate drugs such as risedronate act by inhibiting farnesyl diphosphate synthase, thereby disrupting protein prenylation in osteoclasts. We recently found that an anti-resorptive phosphonocarboxylate analogue of risedronate, 3-PEHPC (previously referred to as NE10790), selectively prevents prenylation of Rab GTPases in vitro by specifically inhibiting Rab geranylgeranyl transferase. In this study, we demonstrate that unprenylated Rab6 could be detected in J774 cells after treatment with 3-PEHPC or risedronate for as little as 4 h, and reached 50% after 24 h.

A role for Rab27b in NF-E2-dependent pathways of platelet formation.

Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology.

Rab GTPases, intracellular traffic and disease.

Membrane and protein traffic in the secretory and endocytic pathways is mediated by vesicular transport. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to human disease. Mutations in Rab27a result in Griscelli syndrome, caused by defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in Tcells.

Polypurine tract formation by Ty1 RNase H.

To better understand the mechanism by which Ty1 RNase H creates the polypurine tract (PPT) primer, we have demonstrated the polymerase-dependent hydrolytic activity of Ty1 reverse transcriptase (RT) during minus-strand synthesis. Using RNase H and polymerase mutants of the recombinant Ty1 RT protein, we show that the two domains of Ty1 RT can act independently of one another. Our results indicate that RNA/DNA substrates containing a short RNA PPT, which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage.

Rab27a is required for regulated secretion in cytotoxic T lymphocytes.

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash).

Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis.

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins.

Fidelity of retrotransposon replication.

Ty1, the genetically tractable retrotransposable element found in the yeast Saccharomyces cerevisiae, closely resembles vertebrate retroviruses both in structure and in mechanism of replication. By direct sequence analysis, we examined the rate and spectrum of new mutations appearing during a single cycle of Ty1 replication. The rate of new mutations was comparable to those seen for replicating retroviruses.

In vivo Ty1 reverse transcription can generate replication intermediates with untidy ends.

Ty1 retrotransposition, like retroviral replication, is a complex series of events requiring reverse transcription of an RNA intermediate, RNA-primed minus- and plus-strand DNA synthesis, multiple strand transfers, and precise cleavages of the template and primers by RNase H. In this report, we examine the structure of in vivo Ty1 replication intermediates, specifically with regard to the behavior of reverse transcriptase upon reaching template ends and to the precision with which RNase H might generate these ends. While the expected 3' termini were always identified, terminal nontemplated bases were also observed at all of the RNA and DNA template ends examined.

Replication errors during in vivo Ty1 transposition are linked to heterogeneous RNase H cleavage sites.

We previously identified a mutational hotspot upstream of the Ty1 U5-primer binding site (PBS) border and proposed a novel mechanism to account for this phenomenon during Ty1 replication. In this report, we verify key points of our model and show that in vivo RNase H cleavage of Ty1 RNA during minus-strand strong-stop synthesis creates heterogeneous 5' RNA ends. The preferred cleavage sites closest to the PBS are 6 and 3 bases upstream of the U5-PBS border.

Replication infidelity during a single cycle of Ty1 retrotransposition.

Retroviruses undergo a high frequency of genetic alterations during the process of copying their RNA genomes. However, little is known about the replication fidelity of other elements that transpose via reverse transcription of an RNA intermediate. The complete sequence of 29 independently integrated copies of the yeast retrotransposon Ty1 (173,043 nt) was determined, and the mutation rate during a single cycle of replication was calculated.

Impact of premature stop codons on mRNA levels in infantile Sandhoff disease.

Sandhoff disease is an autosomal recessive lysosomal storage disease resulting from mutations of the HEXB gene encoding the beta subunit of beta-hexosaminidase A. Fibroblast lines from four patients with the infantile form of the disease were investigated for mutations by single strand conformation polymorphism analysis and direct sequencing of PCR products. Two of the cell lines were homozygous for a common, 16 kb deletion of the 5' end of HEXB gene.

The presence of two different infantile Tay-Sachs disease mutations in a Cajun population.

A study was undertaken to characterize the mutation(s) responsible for Tay-Sachs disease (TSD) in a Cajun population in southwest Louisiana and to identify the origins of these mutations. Eleven of 12 infantile TSD alleles examined in six families had the beta-hexosaminidase A (Hex A) alpha-subunit exon 11 insertion mutation that is present in approximately 70% of Ashkenazi Jewish TSD heterozygotes. The mutation in the remaining allele was a single-base transition in the donor splice site of the alpha-subunit intron 9.

A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: implications for carrier screening.

Deficiency of beta-hexosaminidase A (Hex A) activity typically results in Tay-Sachs disease. However, healthy subjects found to be deficient in Hex A activity (i.e.

Six novel deleterious and three neutral mutations in the gene encoding the alpha-subunit of hexosaminidase A in non-Jewish individuals.

Initial investigations demonstrated that only 3/34 "Tay-Sachs chromosomes" in 22 unrelated, non-Jewish patients or carriers of some form of GM2-gangliosidosis (7 black and 15 non-Jewish Caucasian) had either of the two mutations commonly found in the Jewish population. To determine the nature and incidence of the alterations in this non-Jewish population we have utilized PCR, single-strand conformation polymorphism analysis and sequencing to detect new mutations in genomic DNA. Fourteen primer sets have been utilized to analyze 80% of the coding region and 23/26 splice sites of the gene coding for the alpha chain of hexosaminidase A.

A mutation common in non-Jewish Tay-Sachs disease: frequency and RNA studies.

Tay-Sachs disease (TSD) is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (Hex A). We have discovered that a Tay-Sachs mutation, IVS-9 + 1 G-->A, first detected by Akli et al. (Genomics 11:124-134, 1991), is a common disease allele in non-Jewish Caucasians (10/58 alleles examined).

Molecular basis of hexosaminidase A deficiency and pseudodeficiency in the Berks County Pennsylvania Dutch.

Following the birth of two infants with Tay-Sachs disease (TSD), a non-Jewish, Pennsylvania Dutch kindred was screened for TSD carriers using the biochemical assay. A high frequency of individuals who appeared to be TSD heterozygotes was detected (Kelly et al., 1975).

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction.

Cancer in relatives of leukemic patients with chromosomal rearrangements at rare (heritable) fragile-site locations in their malignant cells.

The cancer occurrence in relatives (N = 407) of 40 case probands (who had leukemia and rearrangements at the same chromosomal location as at least one of 23 recognized rare [heritable] autosomal fragile sites [Sutherland and Mattei 1987]) was compared both to cancer occurrence in relatives (N = 390) of 40 control probands (who had leukemia or other hematologic illness but no recognized chromosomal rearrangements) and to cancer incidence in the general population of the United States. Fragile-site carrier status was not determined in case or control probands. No significant excess of cancer in case relatives, compared with either control relatives or to general (SEER) population expectancies, was found.

Familial isolated aniridia associated with a translocation involving chromosomes 11 and 22 [t(11;22)(p13;q12.2)].

Isolated aniridia segregated as an autosomal dominant trait in a family with 11 affected members spanning five generations. Four of the eight individuals studied had aniridia associated with glaucoma and cataracts. Cytogenetic studies revealed an apparently balanced reciprocal translocation between chromosomes 11 and 22 [t(11;22)(p13;q12.

Reproductive outcomes of paracentric inversion carriers: report of a liveborn dicentric recombinant and literature review.

An abnormal infant had a dicentric chromosome 14 with an inverted tandem duplication [46,XY,inv dup(14) (pter----q32.3::q24.2----pter)], thus making him trisomic for the proximal two-thirds of chromosome 14.

Two different structural abnormalities of chromosome 13 in offspring of chromosomally normal parents with two fragile sites.

Two siblings were found with different structural abnormalities involving their maternally inherited chromosome 13. The proband exhibited a ring 13 and a small fragment: 46,XX,r(13) (p11q34), +f, while her clinically normal brother carried a dicentric Robertsonian translocation: 45,XY,dic(13;15) (p11;p11). Both parents had normal karyotypes in peripheral blood and skin fibroblasts.