RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia

Occurrence of 13q14 deletions between D13S273 and D13S25 in B-cell chronic lymphocytic leukemia (B-CLL) suggests that the region contains a tumor suppressor gene. We constructed a PAC/cosmid contig largely corresponding to a 380-kb 13q14 YAC insert that we found deleted in a high proportion of B-CLL patients. We found seven genes by exon trapping, cDNA screening and analysis/cDNA extension of known expressed sequence tags. One appeared to originate from another region of 13q. Recent publications have focused on two of the genes that most likely do not have a tumor suppressor role. This study evaluates the remaining four genes in the region by mutation scanning and theoretical analysis of putative encoded products. No mutations suggestive of a pathogenic effect were found. The 13q14 deletions may be a consequence of an inherent instability of the region, an idea supported by our finding of a considerable proportion of AluY repeats. Deletion of putative enhancer sequences and/or genes in the region may result in an inactivation of tumor suppression by a haploinsufficiency mechanism. We conclude that RFP2, c13ORF1, and a chromosome 13-specific ST13-like gene, FAM10A4, are the most likely candidates for such a type of B-CLL TSG.     

Delineation of the minimal region of loss at 13q14 in multiple myeloma

Previous studies have focused on the incidence and prognostic implications of 13q14 deletions in multiple myeloma (MM), but none has sought to delineate the minimal common deleted region (CDR). In an effort to do so, dual-color interphase fluorescence in situ hybridization (FISH) was applied on 82 myeloma cases, initially by use of three probes for 13q14 (RB1, D13S319, and D13S25). Deletions were detected in 29/82 (35.4%) cases, and all except one were monoallelic. Subsequently, contiguous YACs, PACs, and a BAC spanning the 13q14-q21 region were employed for deletion mapping in addition to a 13q telomere probe. Large deletions extending to the 13q34 region were found in 55% of the deleted cases, whereas an additional 13.8% showed loss of both 13q34 and 13q14 regions with retention of 13q21. A CDR of approximately 350 kb was identified at 13q14 with the proximal border approximately 120 kb centromeric from D13S319, encompassing an area rich in expressed sequence tagged sites and containing DLEU1, DLEU2, and RFP2 genes. Direct sequencing of the RFP2 gene revealed no mutations in six patients and four MM cell lines harboring deletions of the CDR. However, a role for RFP2 in the pathogenesis of MM cannot yet be excluded, given that alternative mechanisms such as haploinsufficiency remain possible.     

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.     

Leucine biosynthesis in yeast

Summary Tetrad analysis indicates that -isopropylmalate synthase activity of yeast is determined by two separate genes, designated LEU4 and LEU5. LEU4 is identified as a structural gene. LEU5 either encodes another -isopropylmalate synthase activity by itself or provides some function needed for the expression of a second structural gene. The properties of mutants affecting the biosynthesis of leucine and its regulation suggest that the expression of LEU1 and LEU2 (structural genes encoding isopropylmalate isomerase and -isopropylmalate dehydrogenase, respectively) is controlled by a complex of a-isopropylmalate and a regulatory element (the LEU3 gene product). Similarities and differences between yeast and Neurospora crassa with respect to leucine biosynthesis are discussed.This is Journal Paper No. 9347 of the Agricultural Experiment Station, Purdue University