BRIEF REPORTS 273 tial medium (MEM) supplemented with 10% fetal calf serum Mapping of the Human HPRP3 and and phytohemagglutinin at 37°C for 68 h. The lymphocyte cultures were treated with BrdU (0.18 mg/ml) for 20 h to syn- HPRP4 Genes Encoding U4/U6- chronize the cell population. The synchronized cells were Associated Splicing Factors to washed three times to release the block and recultured for 6 h in a-MEM with thymidine (2.5 mg/ml). Cells were harvested Chromosomes 1q21.1 and 9q31–q33 and slides were prepared using standard procedures, including hypotonic treatment, fixation, and air drying (4). Henry H. Q. Heng,* Anan Wang,† and Jim Hu† , ‡ ,1 For HPRP3 mapping, a full-length cDNA probe was used, * Biology Department, York University, 4700 Keele Street, North York, while for HPRP4 mapping, a genomic clone containing the Ontario M3J 1P8, Canada; †Division of Respiratory Research, Hospital entire gene, which was isolated from a PACs library, was for Sick Children, and ‡Department of Paediatrics, University of Toronto, used, (6). Both probes were biotinylated with dATP using 555 University Avenue, Toronto, Ontario M5G 1X8, Canada the GIBCO BRL BioNick labeling kit at 15°C for 1 and 2 h, respectively. Probes were denatured at 75°C in a hybridiza- Received September 16, 1997; accepted December 10, 1997 tion mix consisting of 50% formamide and 10% dextran sul- fate and human Cot I DNA. Probes were loaded onto the denatured chromosomal slides (2). After overnight hybridiza- tion, slides were washed in 50% formamide and 21 SSC Removal of introns from nuclear pre-mRNAs is carried out washing solution. FISH signals were then detected with on an RNA – protein complex called the spliceosome, which FITC-conjugated avidin. For chromosomal identification, consists of a pre-mRNA, four small nuclear ribonucleoprotein DAPI staining was used immediately after FISH detection (snRNP) particles (U1, U2, U5, and U4/U6), and an undefined (3). After a screening under the microscope to check the hy- number of transiently associated splicing factors (7, 10). U4/ bridization efficiency, FISH signals and the DAPI-banded U6 snRNP enters the spliceosome together with U5 snRNP chromosomes were photographed separately, and the assign- immediately before the first step of the splicing reaction. ment of FISH mapping data with chromosomal bands was Upon entering the spliceosome, U4 snRNP dissociates from obtained by superimposing FISH signals with DAPI-banded U6 snRNP which then interacts with U2 snRNP (7, 10). It is chromosomes. currently not clear how these conformational rearrangements The gene encoding HPRP3 maps to chromosome 1q21.2 occur and how they are regulated. It is conceivable that splic- (data not shown) as determined by data summarized from ing factors may contribute to the regulation of these events, 10 photographs. Ninety-two of 100 mitotic figures checked since a number of proteins have been characterized as puta- exhibited signals on one pair of chromosomes. No other sig- tive RNA helicases that might mediate structural rearrange- nificant fluorescent signals were detected on other chromo- ments in the spliceosome (7, 10). The presence of the essential somes (Fig. 1). WD domain in yeast U4/U6 splicing factor Prp4p (1, 5) and The gene encoding HPRP4 maps to chromosome 9q31 – q33 its genetic interaction with another splicing factor, Prp3p (8), (data not shown), based on the analysis of photographs from also suggest that G-protein-like interactions may be involved 10 mitotic figures. Ninety-five of 100 mitotic figures checked these events. showed signals on chromosome 9. No other significant FISH We have recently identified and partially characterized two signals were specifically detected, even though this PAC clone human splicing factors (11). Hprp3p (Genbank Accession No. appears a little high in GC content, which generated minor AF001947), which is present in human U4/U6rU5 tri-snRNP background on chromosome 19 (Fig. 2). These data suggest and more tightly associated with U4/U6 snRNP, is a human that the locus of HPRP4 is only on chromosome 9. homologue of the yeast U4/U6-associated splicing factor, Prp3p. Hprp4p (Genbank Accession No. U82756) is homolo- gous to the yeast U4/U6-associated protein, Prp4p. We have ACKNOWLEDGMENTS shown that Hprp4p coimmunoprecipitates with Hprp3p and interacts with Hprp3p in vitro. The sequence similarity be- We thank Jack Huizenga for isolation of the HPRP4 genomic clone. This work was supported by a Connaught New Staff Matching Grant tween human Hprp4p and yeast Prp4p led to the discovery from University of Toronto and a block term grant from the Ontario of two additional WD repeats in yeast Prp4p. We have also Thoracic Society to J.H. shown that the WD domains of these two proteins are likely folded into a seven-blade propeller structure (11). Human cDNA clones encoding these two splicing factors have also REFERENCES been independently isolated and characterized by others (9). To determine the chromosomal localizations of these two 1. Dalrymple, M. A., Bjørn, S. P., Friesen, J. D., and Beggs, J. D. novel genes, fluorescence in situ hybridization (FISH) was car- (1989). The product of the PRP4 gene of S. cerevisiae shows ried out on human lymphocyte chromosomes. Lymphocytes homology to b subunits of G proteins. Cell 58: 811–812. isolated from human blood were cultured in a-minimal essen- 2. Heng, H. H., Squire, J., and Tsui, L. C. (1992). High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. USA 89: 9509 – 9513. Sequence data from this article have been deposited with the Gen- Bank Data Library under Accession Nos. AF001947 and U82756. 3. Heng, H. H., and Tsui, L. C. (1993). Modes of DAPI banding and simultaneous in situ hybridization. Chromosoma 102: 325– 1 To whom correspondence should be addressed. Telephone: (416) 813-6412. Fax: (416) 813-5771. E-mail: jhu@sickkids.on.ca. 332. GENOMICS 48, 273–275 (1998) ARTICLE NO. GE975181 0888-7543/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.