- Email: [email protected]
The gene cluster for human U2 RNA is located on chromosome 17q21
Experimental Cell Research 159 (1985) 473--478
The Gone Cluster for Human U2 RNA is Located on Chromosome 17q21 KARIN HAMMARSTROM, BERTA SANTESSON, G U N N A R WESTIN and U L F PETTERSSON Department of Medical Genetics and Microbiology, Biomedical Center (BMC), S-75123 Uppsala, Sweden
The gone cluster for human U2 RNA has been mapped to chromosome 17q21 by in situ hybridization and hybridization analysis of DNA from mouse/human somatic cell hybrids. © 1985AcademicPress, Inc.
The human genome contains 20-40 genes encoding U2 RNA [24, 27]. In addition to these bona fide genes a large number of pseudogenes have been detected which appear to have been dispersed throughout the genome by different evolutionary mechanisms [2, 5, 23, 25, 26]. The functional gone copies are present in one or a few clusters consisting of 6.2 kilobase pairs (kbp) long tandem repeats [24, 27]. The human genome also contains a large number of gone copies for U1 R N A which have been mapped to the short arm of chromosome 1 [10, 16]. In the present communication we have determined the chromosomal location of the genes for human U2 RNA. Probes, derived from a human U2 gene were used for in situ hybridization to metaphase chromosomes and for Southern blot analysis of DNA from somatic cell hybrids. The results show that the gone cluster for human U2 R N A is located on chromosome 17 q21.
MATERIALS AND METHODS
In Situ Hybridization Chromosome preparations were obtained from PHA-stimulated normal human male and female cord blood lymphocytes, cultured for 48 or 72 h in RPMI medium supplemented with 20 % fetal calf serum (FCS). BrdU was added to a final concentration of 200 ~tg/ml to synchronize the cells and also to facilitate banding [3, 29]. After 16-17 h the cultures were washed twice and the BrdU block released by incubating the cells in fresh medium containing 10 ~tM thymidine. They were harvested 6--7 h later and hypotonic treatment, fixation and slide preparation were performed according to standard procedures. Elongated, stretched chromosomes of early and mid-metaphases were frequently found, which after staining showed high resolution banding. The chromosomes were G-banded according to Perry & Wolff [18] before in situ hybridization to facilitate identification and the subsequent location of the autoradiographic grains; the slides were stained in Hoechst 33258 (1 ~g/ml in 2xSSC) for 15 min, rinsed with water, then placed in 2 x S S C and exposed to UV light for about 1 h. After rinsing, the slides were stained in 7 % phosphate-buffered Giemsa solution for 15 min, rinsed in water and airdried. Well banded and spread metaphases with a full chromosome complement were photographed. The Giemsa stain was subsequently removed and the slides were further processed for in situ
Exp Cell Res 159 (1985)
474
K. HammarstrOm eta/. 5' ~
U2.6/pBH 1
U2 RNA g e n e
pU2/C I
I
lOObp
Fig. 1. Schematic illustration of the two probes which were used for in situ hybridization and hybridization to DNA from mouse-human hybrids. The subclones were derived from the 6.2 kbp long repeat unit in the gene cluster for human U2 RNA [27]. The box shows the U2 gene and thin lines flanking sequences. Stippled boxes show vector sequences.
hybridization, performed essentially as described by Zabel et al. [29]. After hybridization the slides were dipped in Ilford L4 emulsion, diluted 1 : 1 with distilled water. They were exposed for 8-15 days, developed in Kodak D-19, fixed, and dried and then stained with 7% Giemsa solution in Scirensen phosphate buffer. All photographed cells that had a satisfactory technical quality were scored for grains. Only grains which were located on the chromosome or touching at least one chromatid were counted. The relative distribution of grains was also corrected for the DNA contents of the chromosome arms [13] and corrections were made for the X- and Y-chromosomes, since those were numerically underrepresented.
Hybridization Probes Probes, containing sequences from the human U2 gene cluster (fig. 1) were 3H- or 32p-labelled by nick translation [19].
Somatic Cell Hybrids The somatic cell hybrids originated from fusion of unstimulated normal human lymphocytes and fibroblasts from thymidine kinase deficient mouse L cells, using inactivated Sendal virus as the fusion agent. Hybrid cells were selected and cloned in HAT medium [9], but were later transferred to standard medium. Two different subclones designated 334 and 355 were analysed. They had been frozen in their approx. 30th passage at a time when the total chromosome number was 50-54 including the human chromosome 17 and the essentially unchanged mouse genome of which one chromosome is dicentric and 7 or 8 chromosomes are bi-armed. After thawing, the cells were grown in standard medium for a week before checking the karyotype and extraction of DNA. Analysis of 50 metaphases from each subclone revealed that 90 % of the metaphases in subclone 355 and only 25% in subclone 334 still contained the human chromosome 17.
Southern Blot Hybridization DNA was cleaved with Hind III and separated by electrophoresis in a 0.8 % agarose gel. The fragments were transferred to nitrocellulose according to the method described by Southern [22]. After hybridization the filters were washed in 2xSSC, 0.5% SDS for several hours at 65°C.
RESULTS
Mapping of Human U2 Genes by In Situ Hybridization The results of the in situ hybridization showed a high concentration of grains on chromosome 17 band q21 (fig. 2A, B) and almost all grains on chromosome 17 were within this band, often in q21.3 at the border to q22 (fig. 2B, C). Other chromosome arms which gave a slightly higher number of grains than the background were also analysed in detail. However, in these cases the grains were uniformly distributed along the arms. In some cells, which had been exposed for 15 days confluent grains could sometimes be seen on both chromosomes 17q21 Exp Cell Res 159 (1985)
Localization of human U2 genes 475
!
25
13 12 11.2 11.1 11.I 11.2 lz 21.1 21.2 21.3
20
15
10
¸
oo
ii~
| ::~iii¸
22 23
24 25
Pq Pq pq Pq pq pq Pq pq pq pq pq pq Pq pq pq Pq pq Pq Pq 1 2 3 4 5 6 T 8 9 10111213141516171819202122
pq x y
c
Fig. 2. (A) Distribution of silver grains over human chromosomes as determined by in situ hybridization with a human U2 probe. 44 metaphases were scored for grains. Most of the grains were located on the long arm of chromosome 17 (band q21). (B) Diagram of chromosome 17 indicating the relative positions of the silver grains which were found over chromosome 17. (C) Chromosome 17 from five different metaphases after in situ hybridization with the U2 probe. The picture shows the chromosome before application of the autoradiographic film and after development of the film. Silver grains are located within band q21.
(data not shown). These cells were not included in the analysis on which the histogram in fig. 2A is based, because it is difficult to distinguish these clustered grains from autoradiographic artefacts.
Mapping of Human U2 Genes by the Use of Somatic Cell Hybrids Since human/mouse somatic cell hybrids were available which exclusively contain the human chromosome 17 in a background of mouse chromosomes, it was possible to confirm the chromosomal assignment by Southern blot analysis. The somatic cell hybrids originated from fusion of unstimulated normal human lymphocytes and fibroblasts from thymidine kinase-deficient mouse L cells. Hybrid cells were selected and cloned in HAT medium [9]. Cells which carried human chromosome 17 were selected under these conditions, since this chromosome is known to contain the thymidine kinase locus [15]. It has previously been shown that the 10-20 genes per haploid genome for human U2 RNA are organized in one or a few clusters consisting of 6.2 kbp long tandem repeats [24, 27]. Restriction enzymes which cut this repeat once, such as Hind III, thus give rise to a 6.2 kbp long fragment which can be revealed by Southern blot hybridization, using a U2-specific probe. Analysis of D N A from the human/mouse cell hybrids also revealed the characteristic 6.2 kbp long band (fig. 3), whereas the mouse parental cell line did not show any band at that position. The band observed with D N A from subclone 334 was weaker than that observed with D N A from subclone 355, because there was less than one copy of Exp Cell Res 159 (1985)
476 K. H a m m a r s t r 6 m et al. A
B
C
Fig. 3. Southern blot analysis of approx. 10 Ixg of DNA from human/mouse somatic cell hybrids which
exclusively contain human chromosome 17. The probe was U2,6/pBH1. Two different subclones designated 334 and 355 were analysed which had been frozen in their approx. 30th passage. After thawing, the cells were grown in standard medium for a week before checking the karyotype and extraction of DNA. Approx. 90 % of the metaphases in subclone 355 and only 25 % in subclone 334 still contained the human chromosome 17. Mouse DNA served as a negative control for the background hybridization to mouse chromosomes. The arrow indicates the position of a 6.2 kbp long fragment, as determined by analysing a clone which contained a single copy of the repeat unit in the U2 gene cluster. A, DNA from clone 355; B, DNA from clone 334; C, DNA from the parental mouse cell line.
human chromosome 17 in these cells (in clone 355 90% of the cells contained a single human chromosome 17, whereas only about 25 % of the cells in clone 334 were positive for this chromosome). It is noteworthy that mouse DNA does not give rise to any prominently hybridizing fragment when analysed with the human U2 probe. This is probably due to the fact that the flanking regions are very poorly conserved, even though mouse and human U2 genes are closely related [27]. Moreover, in the mouse genome the U2 genes are present on eight Hind III fragments of different size, none of which contains more than two U2 gene copies [17]. DISCUSSION Taken together the results demonstrate that the majority of the human U2 genes are located on chromosome 17q21. The size of the whole U2 gene cluster is estimated to be 60-120 kbp [24, 27]. The U2 genes thus only represent a small part of the q21 band, since chromosomal bands are estimated to contain about 104 kbp DNA [21]. The results are in agreement with the previous observation Exp Cell Res 159 (1985)
Localization of human U2 genes
477
that genes for human U2 R N A are located in one or possibly a few clusters [24, 27]. The resolution of the method, however, does not permit the conclusion that all genes for human U2 R N A are located at this chromosomal position; one or a few U2 gene copies could still be located at other chromosonal positions. Moreover, it has been demonstrated that the human genome contains a large number of sequences which are related to but not identical with the sequence of human U2 RNA. These so called pseudogenes are probably dispersed throughout the genome. Lund et al. [11] and Naylor et al. [16] have previously demonstrated that the gene family for human U 1 R N A is located on the short arm of chromosome 1. The present results thus show that the genes for at least two human snRNAs are organized in clusters and also that these clusters are located on different chromosomes. Nucleotide sequence studies and restriction enzyme mapping have demonstrated that the individual genes both for human U1 and U2 RNA are surrounded by highly conserved sequences [I 1, 12]. In the case of U2 each gene is flanked by an approx. 6.2 kbp long sequence and the whole unit is repeated 10-20 times [24, 27]. Moreover, transcription studies using the Xenopus laevis oocyte system have demonstrated that only about 250 nucleotides of 5'-flanking sequences are required for efficient transcription [28]. An interesting question is therefore what the function of the remaining sequences is. Several other genes have been assigned to human chromosome 17 (Human Gene Mapping 7 1984 [7]) and some of these appear to be located very close to the position where the human U2 gene cluster maps. They include the thymidine kinase gene [8, 15], the galactokinase gene [4] and the alfa type-1 collagen gene [1, 6]. Moreover, the site on chromosome 17 which is modified by adenovirus-12 has been mapped to this position [14] as has also the breakpoint in the 15 : 17 translocation in patients with promyelocytic leukemia [20]. Genes for growth hormone, chorionic somatomammotropin as well as variants of these genes and pseudogenes are located at a slight distance from the human U2 genes (q22--q24). The U2 gene cluster should serve as an additional useful marker on chromosome 17. We are grateful to Dr Jon Jonasson for teaching us the method of in situ hybridization, to Mrs Jeanette Backman for excellent secretarial aid, and to Mrs Britt-Marie Carlberg for skilful technical assistance. This investigation was supported by grants from the Swedish Medical Research Council, the Swedish National Board for Technical Development and from the Marcus Borgstrfm Foundation.
Note Added in Proof After this paper was submitted, Lindgren et al. [30] reported the assignment by in situ hybridization of the U2 RNA gene cluster to chromosome 17, bands q21-q22. Their results are compatible with the results described in this paper.
REFERENCES 1. Church, R L, Sundar Raj, N & McDougall, J K, Cytogenet cell genet 27 (1980) 24. 2. Denison, R A, Van Arsdell, S W, Bernstein, L B & Weiner, A M, Proc natl acad sci US 78 (1981) 810. Exp Cell Res 159 (1985)
478
K. HammarstrOm
e t al.
3. Dutrilleaux, B & Viegas-Pequignot, E, Hum genet 57 (1981) 93. 4. Elsevier, S M, Kucherlapti, R S, Nichols, E A, Creagan, R P, Giles, R E, Ruddle, F H, Willecke, K & McDougall, J K, Nature 251 (1974) 633. 5. Hammarstr6m, K, Westin, G, Bark, C, Zabielski, J & Pettersson, U, J mol biol 179 (1984) 157. 6. Huerre, C, Junien, C, Weil, D, Chu, M-L, Morabito, M, Nguyen Van Cong, Myers, J C, Foubert, C, Gross, M-S, Prockop, D J, Boue, A, Kaplan, J-C, de la Chapelle, A & Ramirez, F, Proc natl acad sci US 79 (1982) 6627. 7. Human gene mapping 7. Cytogenet cell genet 37 (1984) nos. 1-4. 8. Kucherlapti, R S, McDougall, J K & Ruddle, F H, Cytogenet cell genet 13 (1974) 108-110. 9. Littlefietd, J W, Exp cell res 41 (1966) 190. 10. Lund; E, Bostock, C, Robertson, M, Christie, S, Mitchen, J L & Dahlberg, J E, Mol cell biol 3 (1983) 2211. I1. Lund, E & Dahlberg, J E, J biol chem 259 (1984) 2013. 12. Manser, T & Gesteland, R F, Cell 29 (1982) 257. 13. Mendelsohn, M L, Mayall, B H, Bogart, E, Moore, D H & Perry, B H, Science 179 (1982) 1126. 14. McDougall, J K, J gen virol 12 (1971) 43-51. 15. Miller, O, Allerdice, P W, Miller, D A, Breg, W R & Migeon, B R, Science 173 (1971) 244. 16. Naylor, S L, Zabel, B U, Manser, T, Gesteland, R & Sakaguchi, A Y, Somatic cell and molecular genetics 10 (1984) 307. 17. Nojima, H & Kornberg, R D, J biol chem 258 (1983) 8151. 18. Perry, P & Wolff, S, Nature 251 (1974) 156. 19. Rigby, P W J, Dieckman, M, Rhodes, C & Berg, P, J mol biol 113 (1977) 237. 20. Rowley, J D, Proc natl acad sci US 74 (1977) 5729. 21. Shows, T B, Naylor, S L & Sakaguchi, A Y, Adv human genet 12 (1982) 341. 22. Southern, E M, J mol biol 18 (1975) 503. 23. Van Arsdell, S W, Denison, R A, Bernstein, L B, Weiner, A M, Manser, T & Gesteland, R F, Cell 26 (1981) 11. 24. Van Arsdell, S W & Weiner, A M, Mol cell biol 4 (1984) 492. 25. - - Nucl acids res 12 (1984) 1463. 26. Westin, G, Monstein, H-J, Zabielski, J, Philipson, L & Pettersson, U, Nucl acids res 9 (1981) 6323. 27. Westin, G, Zabielski, J, Hammarstr6m, K, Monstein, H-J, Bark, C & Pettersson, U, Proc natl acad sci US 81 (1984) 3811. 28. Westin, G, Lund, E, Murphy, J T, Pettersson, U & Dahlberg, J E, EMBO j 3 (1984) 3295. 29. Zabel, B U, Naylor, S L, Sakaguchi, A Y, Bell, G I & Shows, T B, Proc natl acad sci US 80 (1983) 6932. 30. Lindgren, V, Ares, M, Jr, Weiner, A & Francke, U, Nature 314 (1985) 115. Received March 7, 1985
Exp Cell Res 159 (1985)
The Gone Cluster for Human U2 RNA is Located on Chromosome 17q21 KARIN HAMMARSTROM, BERTA SANTESSON, G U N N A R WESTIN and U L F PETTERSSON Department of Medical Genetics and Microbiology, Biomedical Center (BMC), S-75123 Uppsala, Sweden
The gone cluster for human U2 RNA has been mapped to chromosome 17q21 by in situ hybridization and hybridization analysis of DNA from mouse/human somatic cell hybrids. © 1985AcademicPress, Inc.
The human genome contains 20-40 genes encoding U2 RNA [24, 27]. In addition to these bona fide genes a large number of pseudogenes have been detected which appear to have been dispersed throughout the genome by different evolutionary mechanisms [2, 5, 23, 25, 26]. The functional gone copies are present in one or a few clusters consisting of 6.2 kilobase pairs (kbp) long tandem repeats [24, 27]. The human genome also contains a large number of gone copies for U1 R N A which have been mapped to the short arm of chromosome 1 [10, 16]. In the present communication we have determined the chromosomal location of the genes for human U2 RNA. Probes, derived from a human U2 gene were used for in situ hybridization to metaphase chromosomes and for Southern blot analysis of DNA from somatic cell hybrids. The results show that the gone cluster for human U2 R N A is located on chromosome 17 q21.
MATERIALS AND METHODS
In Situ Hybridization Chromosome preparations were obtained from PHA-stimulated normal human male and female cord blood lymphocytes, cultured for 48 or 72 h in RPMI medium supplemented with 20 % fetal calf serum (FCS). BrdU was added to a final concentration of 200 ~tg/ml to synchronize the cells and also to facilitate banding [3, 29]. After 16-17 h the cultures were washed twice and the BrdU block released by incubating the cells in fresh medium containing 10 ~tM thymidine. They were harvested 6--7 h later and hypotonic treatment, fixation and slide preparation were performed according to standard procedures. Elongated, stretched chromosomes of early and mid-metaphases were frequently found, which after staining showed high resolution banding. The chromosomes were G-banded according to Perry & Wolff [18] before in situ hybridization to facilitate identification and the subsequent location of the autoradiographic grains; the slides were stained in Hoechst 33258 (1 ~g/ml in 2xSSC) for 15 min, rinsed with water, then placed in 2 x S S C and exposed to UV light for about 1 h. After rinsing, the slides were stained in 7 % phosphate-buffered Giemsa solution for 15 min, rinsed in water and airdried. Well banded and spread metaphases with a full chromosome complement were photographed. The Giemsa stain was subsequently removed and the slides were further processed for in situ
Exp Cell Res 159 (1985)
474
K. HammarstrOm eta/. 5' ~
U2.6/pBH 1
U2 RNA g e n e
pU2/C I
I
lOObp
Fig. 1. Schematic illustration of the two probes which were used for in situ hybridization and hybridization to DNA from mouse-human hybrids. The subclones were derived from the 6.2 kbp long repeat unit in the gene cluster for human U2 RNA [27]. The box shows the U2 gene and thin lines flanking sequences. Stippled boxes show vector sequences.
hybridization, performed essentially as described by Zabel et al. [29]. After hybridization the slides were dipped in Ilford L4 emulsion, diluted 1 : 1 with distilled water. They were exposed for 8-15 days, developed in Kodak D-19, fixed, and dried and then stained with 7% Giemsa solution in Scirensen phosphate buffer. All photographed cells that had a satisfactory technical quality were scored for grains. Only grains which were located on the chromosome or touching at least one chromatid were counted. The relative distribution of grains was also corrected for the DNA contents of the chromosome arms [13] and corrections were made for the X- and Y-chromosomes, since those were numerically underrepresented.
Hybridization Probes Probes, containing sequences from the human U2 gene cluster (fig. 1) were 3H- or 32p-labelled by nick translation [19].
Somatic Cell Hybrids The somatic cell hybrids originated from fusion of unstimulated normal human lymphocytes and fibroblasts from thymidine kinase deficient mouse L cells, using inactivated Sendal virus as the fusion agent. Hybrid cells were selected and cloned in HAT medium [9], but were later transferred to standard medium. Two different subclones designated 334 and 355 were analysed. They had been frozen in their approx. 30th passage at a time when the total chromosome number was 50-54 including the human chromosome 17 and the essentially unchanged mouse genome of which one chromosome is dicentric and 7 or 8 chromosomes are bi-armed. After thawing, the cells were grown in standard medium for a week before checking the karyotype and extraction of DNA. Analysis of 50 metaphases from each subclone revealed that 90 % of the metaphases in subclone 355 and only 25% in subclone 334 still contained the human chromosome 17.
Southern Blot Hybridization DNA was cleaved with Hind III and separated by electrophoresis in a 0.8 % agarose gel. The fragments were transferred to nitrocellulose according to the method described by Southern [22]. After hybridization the filters were washed in 2xSSC, 0.5% SDS for several hours at 65°C.
RESULTS
Mapping of Human U2 Genes by In Situ Hybridization The results of the in situ hybridization showed a high concentration of grains on chromosome 17 band q21 (fig. 2A, B) and almost all grains on chromosome 17 were within this band, often in q21.3 at the border to q22 (fig. 2B, C). Other chromosome arms which gave a slightly higher number of grains than the background were also analysed in detail. However, in these cases the grains were uniformly distributed along the arms. In some cells, which had been exposed for 15 days confluent grains could sometimes be seen on both chromosomes 17q21 Exp Cell Res 159 (1985)
Localization of human U2 genes 475
!
25
13 12 11.2 11.1 11.I 11.2 lz 21.1 21.2 21.3
20
15
10
¸
oo
ii~
| ::~iii¸
22 23
24 25
Pq Pq pq Pq pq pq Pq pq pq pq pq pq Pq pq pq Pq pq Pq Pq 1 2 3 4 5 6 T 8 9 10111213141516171819202122
pq x y
c
Fig. 2. (A) Distribution of silver grains over human chromosomes as determined by in situ hybridization with a human U2 probe. 44 metaphases were scored for grains. Most of the grains were located on the long arm of chromosome 17 (band q21). (B) Diagram of chromosome 17 indicating the relative positions of the silver grains which were found over chromosome 17. (C) Chromosome 17 from five different metaphases after in situ hybridization with the U2 probe. The picture shows the chromosome before application of the autoradiographic film and after development of the film. Silver grains are located within band q21.
(data not shown). These cells were not included in the analysis on which the histogram in fig. 2A is based, because it is difficult to distinguish these clustered grains from autoradiographic artefacts.
Mapping of Human U2 Genes by the Use of Somatic Cell Hybrids Since human/mouse somatic cell hybrids were available which exclusively contain the human chromosome 17 in a background of mouse chromosomes, it was possible to confirm the chromosomal assignment by Southern blot analysis. The somatic cell hybrids originated from fusion of unstimulated normal human lymphocytes and fibroblasts from thymidine kinase-deficient mouse L cells. Hybrid cells were selected and cloned in HAT medium [9]. Cells which carried human chromosome 17 were selected under these conditions, since this chromosome is known to contain the thymidine kinase locus [15]. It has previously been shown that the 10-20 genes per haploid genome for human U2 RNA are organized in one or a few clusters consisting of 6.2 kbp long tandem repeats [24, 27]. Restriction enzymes which cut this repeat once, such as Hind III, thus give rise to a 6.2 kbp long fragment which can be revealed by Southern blot hybridization, using a U2-specific probe. Analysis of D N A from the human/mouse cell hybrids also revealed the characteristic 6.2 kbp long band (fig. 3), whereas the mouse parental cell line did not show any band at that position. The band observed with D N A from subclone 334 was weaker than that observed with D N A from subclone 355, because there was less than one copy of Exp Cell Res 159 (1985)
476 K. H a m m a r s t r 6 m et al. A
B
C
Fig. 3. Southern blot analysis of approx. 10 Ixg of DNA from human/mouse somatic cell hybrids which
exclusively contain human chromosome 17. The probe was U2,6/pBH1. Two different subclones designated 334 and 355 were analysed which had been frozen in their approx. 30th passage. After thawing, the cells were grown in standard medium for a week before checking the karyotype and extraction of DNA. Approx. 90 % of the metaphases in subclone 355 and only 25 % in subclone 334 still contained the human chromosome 17. Mouse DNA served as a negative control for the background hybridization to mouse chromosomes. The arrow indicates the position of a 6.2 kbp long fragment, as determined by analysing a clone which contained a single copy of the repeat unit in the U2 gene cluster. A, DNA from clone 355; B, DNA from clone 334; C, DNA from the parental mouse cell line.
human chromosome 17 in these cells (in clone 355 90% of the cells contained a single human chromosome 17, whereas only about 25 % of the cells in clone 334 were positive for this chromosome). It is noteworthy that mouse DNA does not give rise to any prominently hybridizing fragment when analysed with the human U2 probe. This is probably due to the fact that the flanking regions are very poorly conserved, even though mouse and human U2 genes are closely related [27]. Moreover, in the mouse genome the U2 genes are present on eight Hind III fragments of different size, none of which contains more than two U2 gene copies [17]. DISCUSSION Taken together the results demonstrate that the majority of the human U2 genes are located on chromosome 17q21. The size of the whole U2 gene cluster is estimated to be 60-120 kbp [24, 27]. The U2 genes thus only represent a small part of the q21 band, since chromosomal bands are estimated to contain about 104 kbp DNA [21]. The results are in agreement with the previous observation Exp Cell Res 159 (1985)
Localization of human U2 genes
477
that genes for human U2 R N A are located in one or possibly a few clusters [24, 27]. The resolution of the method, however, does not permit the conclusion that all genes for human U2 R N A are located at this chromosomal position; one or a few U2 gene copies could still be located at other chromosonal positions. Moreover, it has been demonstrated that the human genome contains a large number of sequences which are related to but not identical with the sequence of human U2 RNA. These so called pseudogenes are probably dispersed throughout the genome. Lund et al. [11] and Naylor et al. [16] have previously demonstrated that the gene family for human U 1 R N A is located on the short arm of chromosome 1. The present results thus show that the genes for at least two human snRNAs are organized in clusters and also that these clusters are located on different chromosomes. Nucleotide sequence studies and restriction enzyme mapping have demonstrated that the individual genes both for human U1 and U2 RNA are surrounded by highly conserved sequences [I 1, 12]. In the case of U2 each gene is flanked by an approx. 6.2 kbp long sequence and the whole unit is repeated 10-20 times [24, 27]. Moreover, transcription studies using the Xenopus laevis oocyte system have demonstrated that only about 250 nucleotides of 5'-flanking sequences are required for efficient transcription [28]. An interesting question is therefore what the function of the remaining sequences is. Several other genes have been assigned to human chromosome 17 (Human Gene Mapping 7 1984 [7]) and some of these appear to be located very close to the position where the human U2 gene cluster maps. They include the thymidine kinase gene [8, 15], the galactokinase gene [4] and the alfa type-1 collagen gene [1, 6]. Moreover, the site on chromosome 17 which is modified by adenovirus-12 has been mapped to this position [14] as has also the breakpoint in the 15 : 17 translocation in patients with promyelocytic leukemia [20]. Genes for growth hormone, chorionic somatomammotropin as well as variants of these genes and pseudogenes are located at a slight distance from the human U2 genes (q22--q24). The U2 gene cluster should serve as an additional useful marker on chromosome 17. We are grateful to Dr Jon Jonasson for teaching us the method of in situ hybridization, to Mrs Jeanette Backman for excellent secretarial aid, and to Mrs Britt-Marie Carlberg for skilful technical assistance. This investigation was supported by grants from the Swedish Medical Research Council, the Swedish National Board for Technical Development and from the Marcus Borgstrfm Foundation.
Note Added in Proof After this paper was submitted, Lindgren et al. [30] reported the assignment by in situ hybridization of the U2 RNA gene cluster to chromosome 17, bands q21-q22. Their results are compatible with the results described in this paper.
REFERENCES 1. Church, R L, Sundar Raj, N & McDougall, J K, Cytogenet cell genet 27 (1980) 24. 2. Denison, R A, Van Arsdell, S W, Bernstein, L B & Weiner, A M, Proc natl acad sci US 78 (1981) 810. Exp Cell Res 159 (1985)
478
K. HammarstrOm
e t al.
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