Mia Horowitz

Prevalent and rare mutations among Gaucher patients

Sequence analysis of gcs cDNA (encoding glucocerebrosidase) or genomic fragments originated from Gaucher patients revealed novel mutations. Two rare mutations were found in a type-2 non-Jewish Gaucher patient: a G----A transition (Gly325----Arg) at nucleotide (nt) 5306 of the active gene and a T----G transversion (Cys342----Gly) at nt 5357. These mutations were not found in any other patient. A G----C transversion (Asp409----His) at nt 5957 was identified in two non-Jewish patients, and was designated TL. Two recombinant alleles were found. One recombinant allele designated recTL contained four single-nt mutations. These mutations included: (1) a G----C transversion at nt 5957 (Asp409----His) (the TL mutation); (2) a T----C transition at nt 6433 (Leu444----Pro) creating a new NciI site (NciI mutation); (3) a G----C transversion at nt 6468 (Ala456----Pro; 456 mutation); and (4) a G----C transversion at amino acid (aa) 460 (nt 6482), not associated with any aa change. Sequence analysis indicated that at least part of exon 9, intron 9 and exon 10 of the recombinant gene derived from the pseudogene. The other recombinant gene, designated recNciI, contained a mutation at aa 444 (NciI mutation), and mutations 456 and 460 described above; at least exon 10 of this gene originated from the pseudogene. We hypothesize that the presence of the pseudogene close to the active gene causes transfer of mutations into the active gene via gene conversion or nonhomologous recombination, thus accounting for the high frequency of mutations observed in the gcs gene.

Three unique base pair changes in a family with Gaucher disease.

SummarySingle-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp140→His), an A to C transversion at nucleotide 3170 (Lys157→Gln) and a G to A change at nucleotide 5309 (Glu326→Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.

The metabolism of SV40 RNA is associated with the cytoskeletal framework

Abstract We prepared the cytoskeletal framework of SV40-infected BSC-1 cells late after infection by extracting the cells with Triton X-100. The cytoskeletal framework obtained by this procedure carries more than 80% of the newly synthesized viral polyribosomes. Almost all the viral-specific cytoplasmic RNA is engaged in the formation of cytoskeletal-associated polyribosomes. The cytoskeletal framework harbors both the poly(A) + 19 S and 16 S late viral RNAs, as well as most of the poly(A) − viral RNA. The poly(A) − viral RNA sediments in sucrose gradients between 4 and 18 S representing heterogeneous species and comprises about 30–50% of the total virus-specific RNA in the cytoplasm. Based on pulse-chase experiments it is concluded that: (i) the poly(A) + viral RNA emerges from the nucleus and becomes associated with the cytoskeletal framework, (ii) the decay rate of the poly(A) + RNA species on the cytoskeletal framework is faster than that of the poly(A) − viral RNA, and (iii) both the poly(A) + and poly(A) − viral RNAs detach from the cytoskeletal framework and move to the soluble fraction.

Differential expression of the human glucocerebrosidase-coding gene

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.

The initiation of transcription of sv40 DNA at late time after infection

Abstract In vivo labeled RNA was purified from productively infected cells and in vitro labeled RNA was purified from transcriptional complexes of SV40. The purified RNAs were denatured and fractionated by sedimentation through sucrose gradients. Labeled RNAs of various lengths were hybridized with restriction fragments of SV40 DNA of a known order. In both cases the shortest RNAs hybridized with a fragment which spans between 0.67 and 0.76 map units and the hybridization with this fragment decreased with successively longer RNAs indicating that transcription initiates within this fragment or very close to it. Similar enrichment for this fragment was obtained using nascent RNA chains labeled in vitro with a short pulse. Electron microscopic analysis of transcriptional complexes of SV40 has revealed a substantial fraction with one short nascent RNA chain. The initiation site of the nascent chains was mapped at coordinate 0.67 ± 0.02. The accumulation of transcriptional complexes with short nascent chains, initiated at coordinate 0.67 ± 0.02, and the abundance of labeled nascent RNAs complementary to a fragment spanning between 0.67 and 0.76 map units could indicate the existence of an attenuator site in which RNA chain elongation is blocked, unless a stimulating factor is present which allows transcription to continue into a complete transcript.

Characterization of the Normal Human Glucocerebrosidase Genes and a Mutated Form in Gaucher’s Patient

Gaucher’s disease is the most prevalent lysosomal disease1 It is due to the defective activity of the lysosomal enzyme β-acid glucosidase (glucocerebrosidase E.C.3.2.1.45). Based on clinical signs including presence and severity of neuronopathic involvement it has been divided into three major prototypes: Type I is the non-neurcnopathic form and Types II and III are the neuronopathic forms2,3. All three forms of the disease appear to be caused by rotations of the same gene, since complementation cannot be demonstrated4,5.

Structure and activity of the translocated c-myc in mouse plasmacytoma XRPC-24

In the mouse plasmacytoma XRPC-24 both c-mos and c-myc are rearranged. We cloned the rearranged c-myc and found that it was translocated to the immunoglobulin C alpha locus. The breakpoint is at the end of exon 1 in c-myc and approximately 0.5 kb upstream from exon 1 of C alpha. The cloned translocated c-myc linked to a strong transcriptional promoter can efficiently transform rat embryo fibroblasts when co-transfected with the activated Ha-ras. The transformed cells are tumorigenic in syngeneic rats.