Brief Communication
A ribosomal protein-lke sequence in the 3’ ultranslated region of the HLA-F gene U
J. Zemmour, P. Parham. A ribosomal protein-like sequence in the 3’ untranslated region of the HLA-F gene. Tissue Antigens 1992: 40: 250-253. 0Munksgaard 1992
Jacqueline Zemmour and Peter Parham Departments of Cell Biology, and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
Key wards class I HLA-F - S’UTR - ribosomal proteins - sequence similarity
Received 7 July, accepted for publication 29 July 1992
In addition to the serofogically-defined HLA-A,B and C genes, the HLA region contains a number of structurally-related class I genes. Of these, HLAE, F and G are intact and potentially functional class I genes. They exhibit low polymorphism, are expressed with tissue specificity and their functions are poorly understood (1). Gene transfer studies have shown that HLA-E, F and G each encode a class I heavy chain that associates with P2-microglobulin; however, only HLA-G is transported to the cell surface and its expression is restricted to the cytotrophoblast (3,3, 4). In contrast, HLA-E transcription has been detected in lymphoid and non-lymphoid tissues (5) and HLA-F transcripts have been found in resting T cells, skin, EpsteinBarr virus-transformed B-cell lines, but not in liver, fibroblasts, T-cell lines or the myelomonocytic line HL-60 (6, 7). Although the exon-intron organization of HLAE, F and G is similar to that of HLA-A,B and C, there are features that distinguish these genes in their 3’ regions. HLA-G has a stop codon in exon 6 encoding the transmembrane domain, whereas HLA-E has insertion of 3 Alu sequences combined with a 5bp deletion in exon 7, resulting in translation termination in that exon. Perhaps most distinctive is HLA-F (previously called HLA-5.4) which is the most telomeric class I gene in the HLA region (8). This gene has an altered splice site which results in the elimination of exon 7 , a feature preserved in the chimpanzee homologue of HLA-F (9), and an unusual 3’ untranslated region. Previously, Geraghty et al. ( 6 ) also found that “The 3’ untranslated region of HLA-5.4 is distinct from that of ail other class I genes. The first 32 bp in this region are homologous to other class I genes 250
while the remainder of this sequence diverges completely.” Furthermore, in screening the available sequence banks, Geraghty et al. found no significant matches with other genes. Whilst aligning the class I HLA genes in an analysis of HLA-J (10) we encountered this unusual sequence, which extends from positions 3826-4316 in the 3’ untranslated region of the HLA-F gene. In addition this sequence contains two poly A addition signals 90 bp downstream from the start of the 3’UTR (Fig. 1). When this entire sequence was used to search for sequence homologies, none was found. However, when the sequence was divided a significant homology between nucleotides 3997-43 16 and proteins of the large ribosomal subunit from a number of species was observed. This came from searching the Genbank and EMBL data bases, with the fragment 395 1-4316, using the Fasta program (provided by the GCG package) (1 1) (Fig. 2). Highest homology
3uTR Of HLA-F
-+
-C RiboMul prcin orientation
C I p u l O l i ~ ~
FulyA
FulyA
& j Ri~pocein-wrc
3792
3826 13597
I I I I
I I
3951
I
Segmentsuhidmwb
4316
Figure 1. Schematic representation of the 3’ untranslated region of HLA-F. Numbers indicate nucleotide positions in the sequence. Boxes show the region of the sequence that is homologous to other class I HLA genes or to the ribosomal proteins.
Brief Communication
50 1 MAPSAKATAA KKAVVKGTNG KKALKVRTSA TFRLPKTLKL ARRPKYASKA YSc L 2 5 MAPSTKAASA KKAVVKGSNG SKALKVRTST TFRLPKTLKL TRAPKYARKA YCu L 2 5 Mva L 2 3 Hma L 2 3 HLA-F Wheat L 2 3 Rice L 2 3 Maize L 2 3 Mpo L23 E u g r L23 E . C o l i L23 Y p s L23 Mca L 2 3 B s t L23
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
YSc L25 YCu
Mva L 2 3
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
:...... EE&
.--.:&:: ....
Hma L 2 3 HLA-F Wheat L 2 3 M R i c e L23 M Maize L 2 3 M Mpo L23 M Eugr L 2 3 M F Y F I S R K F Y E . C o l l L 2 3 ..MIREERLL Y p s L23 .MIREERLL Mca L 2 3 MHIT Bst L 2 3 .M@R
.........
......... ......... .........
.
...... ....
101
Hma L 2 3
............. ............. ............ ............ ...........
Wheat L 2 3
RIGPILGHTM H
Maize L 2 3
RMGPILGHTM KIGTTTGYTV RSNNFEGMKK
LFF L 2 3 LE'F E u g r L23 TAF E.C.011 L 2 3 KLF
MpO
150
.PGYSIPLLD
RETN.
RLGKWGKXP Figure 2. Alignment of the ribosomal protein-related sequences with the 3' ribosomal-like protein sequence of HLA-F. Boxed residues indicate identity between HLA-Fand the considered sequence. Sequences are from yeast: YSC, Succharomyces cnrlsbergensis (12)- YCU,Candidu utilis (13); Archuebacteria: Mvo, Methanococcus vannieli (14), Hma, Halobacteriwn mnrismortui (15); Plants: What, Triticwn aestivum (16), Rice, Orpa sutiva (17), Maize, Zea mays (18). Mpo. Marchuntia polyrnorpha (Liverwort) (19). Eugr, euglena gracilis (20);Eubacteria: E . Coli, Escherichiu coli ( 2 I), Yps, Yersinia pseudotuberculosis (22), M a . Mycoplrrsma capricolum (231, Bst, Bacillus stearothermophilur (24).
was found with the L25 protein of the yeast candida utilis, for which there is 60.5% sequence identity in the 329 bp region encompassing nucleotides
3997-4316. This results in 73% similarity or 54% identity of their deduced protein sequences. There are also significant similarities with the L23 protein 25 1
Zemmour & Parham
from bacteria and plants. Interestingly, the sequence from the HLA-F gene is more closely related to the ribosomal protein of yeast and archaebacteria than to those of plants and eubacteria (Fig. 3). Homologues of these ribosomal proteins in higher eukaryotes have not been previously described. The orientation of the reading frame for the ribosomal protein-like sequence is reversed from that of HLA-F (Fig. 1) and the sequence corresponds to the carboxy-terminal 87 residues of the 142 residue of the L25 protein. The sequence of HLA-F that is 3' to this region has yet to be sequenced, and thus it is not known,whether a sequence homologous to the entire L25 gene is represented in HLA-F. There is no evidence as to whether the ribosomal protein-like sequence that has been inserted into HLA-F is a processed or non-processed gene because the yeast L25 gene has no intronic sequence in the region homologous to HLA-F. Furthermore, it is possible that the unknown segment joining the HLA sequence to the ribosomal protein-like sequence (Fig. 1) could contain regulatory signals that may regulate the gene expression of the ribosomal protein. This junctional sequence contains two
c
Candida ufilu
Melhanocaccur vanniclii
Halobacnn~umman'smorlui --
poly A sites, one of which could belong to the ribosomal protein gene. The similarity of the 3' untranslated region of HLA-F with prokaryotic sequences raised the possibility that the presence of the ribosomal protein-like sequence could be an artefact of cloning. This was ruled out by the following experiment. A 3' oligonucleotide primer derived from the ribosome-related sequence was combined with a 5' primer derived from the class I-related sequence to amplify genomic DNA from different human Bcell lines. The polymerase chain reaction was performed in a volume of 100 pl, using 10 p1 of genomic DNA and 90 pmoles of each primer; HLA-F5p frGT GAC TGA TAC GAA TTT GTT CAT GI, HLA-F3p WC GAC TGG CTC CTG ACT ACG ATG C]. The amplification procedure consisted of 35 cycles involving a denaturation step of 1 min at 94"C, an annealing step of 1 min at 67°C and an extension step of 1 min at 72°C. Amplification products were then analyzed by gel electrophoresis on a 2% agarose gel. From the sequence of the cloned HLA-F gene this amplification was predicted to give a 365 bp fragment. Such a fragment was indeed the major product seen in the amplification from all 6 cell lines, showing that the ribosomal protein-like sequence present in the cloned HLA-F gene is not the result of a cloning artefact (Fig. 4). As a control the HLA-FSp primer was used in conjunction with the HLA-3pC primer that we have used for amplification of HLA-A,B,C cDNAs (25). In this case none of the 365 bp fragment was seen; instead, an expected fragment of 256 bp was obtained. As further controls, use of "a single oligonucleotide primer in the polymerase chain reaction with either HLA-F5p or HLA-F3p did not lead to DNA amplification. Using a probe derived from the 3' untranslated
Oryu ruriva
1 2 3 4 5 6 7 8 9101112
L
-
E $ c h e W h Coli
-
-
252
Eaglenu graeilia
Ycninia prerrrdolubrrcrlosu
M J C O P ~ eapricolurn M
Figure 4. PCR amplification of genomic DNA derived from the B cell lines; JY, T7527, LB, LBF, JHAF, IDF, (lanes 1 to 6 respectively) using HLA-FSp and HLA-F3p primers. Lanes 7-8 show amplified products obtained from the JY (lane 7) and T7527 (lane 8) using the HLA-F5p and HLA-3pC primers. PCR amplification using a single primer, either HLA-F5p (JY: lane 9; T7.527: lane 10) or HLA-F3p (JY: lane 1I, T7527: lane 12).
Brief Communication region of HLA-F for Southern blot analysis, Geraghty et al. found 30-50 bands under conditions of low stringency compared to the single HLA-F band revealed under conditions of high stringency (6). That probe is now known to include the ribosomal protein-like sequence. This indicates, first, that there are many sequences related to the yeast L2.5 ribosomal protein in the human genome and, second, that the sequence found in the HLA-F gene is likely to be a unique variant of this sequence. From northern blot analysis, Geraghty et al. showed that some of these sequences were expressed in many tissues, suggestingthat they corresponded to “housekeeping genes” of which those encoding ribosomal proteins would be classic examples. A transcript of 600 bp was particularly prominent and is of sufficient size to encode a ribosomal protein like L25. However, the functional significance of the ribosomal-like sequence in the 3‘ untranslated region of HLA-F, and whether it is transcribed independently of the class I-like sequences of this gene, are questions that have yet to be answered. Acknowledgments
This research was supported by a Shannon Award (A1 242.58) from the NIH. References I . Heinrichs H, Orr HT. HLA non-A,B,C class I genes: their structure and expression. Immunol Res 1990: 9: 265-74. 2. Shimizu Y, Geraghty DE, Koller BH, Orr HT. DeMars R. Transfer and expression of three cloned human non-HLAA,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc Natl Acad Sci USA 1988: 85: 227-31. 3. Ellis SA, Palmer MS, McMichael AJ. Human trophoblasts and the choriocarcinorna cell line BeWo express a truncated HLA class I molecule. J Imrnunol 1990: 144: 731-5. 4. Kovats S, Main EK, Librach C, Stubblebire M, Fisher SJ, DeMan R. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990: 248: 220-3. 5. Wei X, Orr HT. Differential expression of HLA-E, HLAF, and HLA-G transcripts in human tissue. Hwn Immunol 1990: 29: 131-42. 6. Geraghty DE, Wei X, Orr HT, Koller BH. Human leukocyte antigen F (HLA-F). An expressed HLA gene composed of a class I coding sequence linked to a novel transcribed repetitive element. J Exp Med 1990: 171: 1-18. 7. Lury D, Epstein H, Holmes N. The human class I MHC gene HLA-F is expressed in lymphocytes. Int Immunul1990: 2: 531-7. 8. Vemet C, Chimini G, Boretto J, Le Bouteiller P,Pontarotti P. Organization and Evolution of the MHC Chromosomal Region: An Overview. In: Klein J, Klein D. eds. Molecular Evolution of the Major Histocompatibility Complex, Berlin, Heidelberg: Springer-Verlag, 1991: 1-1 1. 9. Lawlor DA, Ward FE. Ennis PD, Jackson AP, Parham P. HLA-A and B polymorphism predate the divergence of humans and chimpanzees. Nature 1988: 335: 268-71.
10. Messer G, Zemmour J, Orr HT, Parham P, Weiss EH,
Girdlestone J. HLA-J, a second inactivated class I HLA gene related to HLA-G and HLA-A. Implications for the evolution of the HLA-A-related genes. J Immunol 1992: 148: 4043-53. 11. Devereux J, Haeberli P, Smithies 0. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984: 12: 387-95. 12. Leer RJ, Van Raamsdonk-Duin MMC, Hagendoom MJM, Mager WH, Planta RJ. Structural comparison of yeast ribosomal protein genes. Nucleic Acids Res 1984: 12: 6681-700. 13. Woudt LP, Mager WH, Beek JG, Wassenaar GM, Planta RJ. Structural and putative regulatory sequences of the gene encoding ribosomal protein L2S in Can&& utilir. Curr Genet 1987: 12: 193-8. 14. Kijpke AKE, Wittmann-Liebold B. Sequence of the gene for ribosomal protein L23 from the archaebacterium Merhanococcus vannielii. FEBS Letters 1988: 239: 313-8. 15. Arndt E, Krijmer W, Hatakeyama T. Organization and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the Archaebacterium Halobacteriwn marismortui. J Biol Chem 1990: 265: 3034-9. 16. Bowman CM, Barker RF, Dyer TA. In wheat ctDNA, segments of ribosomal protein genes are dispersed repeats, probably conserved by nonreciprocal recombination. Curr Genet 1988: 14: 127-36. 17. Hiratsuka J, Shimada H. Whittier R, et al. The complete sequence of the rice (Oryza saliva) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mu1 Gen Genef 1989: 217: 185-94. 18. McLaughlin WE, Larrinua IM. The sequence of the maize plastid encoded rpl 23 locus. Nucleic Acids Res 1988: 16: 8183. 19. Fukuzawa H, Kohchi T, Sano T. et al. Structure and organization of Marchantia polymorpha chloroplast genome. 111 Gene organization of the large single copy region from rbcL to trnl (CAU). J Mol Biol 1988: 203: 333-51. 20. Christopher DA, Cushman JC. Price CA, Hallick RB. Organization of ribosomal protein genes rp123, rpll2, rpsl9, rp122 and rps3 on the Euglena grucilis chloroplast genome. Citrr Genef 1988: 14: 275-86. 21. Zurawski G, Zurawski SM. Structure of the Eschericin coli SIO ribosomal protein opcron. Nucl A c i d Res 1985: 13: 4521-6. 22. Gross U, Chen J-H, Kono DH, Lob0 JG, Yu DTY. High degree of conservation between ribosomal proteins of Yersinia pseudotuberculosis and Escherichiu colt. Nucleic Acids Res 1989: 17: 3601-2. 23. Ohkubo S, Muto A, Kawauchi Y,Yamao F, Osawa S . The ribosomal protein gene cluster of Mycoplasma capricvlum. Mol Gen Genet 1987: 210: 314-22. 24. Kimura M, Kimura J, Ashman K. The complete primary structure of ribosomal proteins LI, L14, L15. L23, L24 and L29 from Bacillus stearothermophilus. Eur J Biochem 1985: 150: 491-7. 25. Zemmour J. Little A-M, Schendel DJ, Parham P. The HLA-A,B. “Negative” mutant cell line C l R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J Immwol 1992: 148: 1941-8. Address: Peter Parham Depts. of Cell Biology and Microbiology and Immunology Stanford University School of Medicine Stanford, CA 94305 U.S.A.
253
A ribosomal protein-lke sequence in the 3’ ultranslated region of the HLA-F gene U
J. Zemmour, P. Parham. A ribosomal protein-like sequence in the 3’ untranslated region of the HLA-F gene. Tissue Antigens 1992: 40: 250-253. 0Munksgaard 1992
Jacqueline Zemmour and Peter Parham Departments of Cell Biology, and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
Key wards class I HLA-F - S’UTR - ribosomal proteins - sequence similarity
Received 7 July, accepted for publication 29 July 1992
In addition to the serofogically-defined HLA-A,B and C genes, the HLA region contains a number of structurally-related class I genes. Of these, HLAE, F and G are intact and potentially functional class I genes. They exhibit low polymorphism, are expressed with tissue specificity and their functions are poorly understood (1). Gene transfer studies have shown that HLA-E, F and G each encode a class I heavy chain that associates with P2-microglobulin; however, only HLA-G is transported to the cell surface and its expression is restricted to the cytotrophoblast (3,3, 4). In contrast, HLA-E transcription has been detected in lymphoid and non-lymphoid tissues (5) and HLA-F transcripts have been found in resting T cells, skin, EpsteinBarr virus-transformed B-cell lines, but not in liver, fibroblasts, T-cell lines or the myelomonocytic line HL-60 (6, 7). Although the exon-intron organization of HLAE, F and G is similar to that of HLA-A,B and C, there are features that distinguish these genes in their 3’ regions. HLA-G has a stop codon in exon 6 encoding the transmembrane domain, whereas HLA-E has insertion of 3 Alu sequences combined with a 5bp deletion in exon 7, resulting in translation termination in that exon. Perhaps most distinctive is HLA-F (previously called HLA-5.4) which is the most telomeric class I gene in the HLA region (8). This gene has an altered splice site which results in the elimination of exon 7 , a feature preserved in the chimpanzee homologue of HLA-F (9), and an unusual 3’ untranslated region. Previously, Geraghty et al. ( 6 ) also found that “The 3’ untranslated region of HLA-5.4 is distinct from that of ail other class I genes. The first 32 bp in this region are homologous to other class I genes 250
while the remainder of this sequence diverges completely.” Furthermore, in screening the available sequence banks, Geraghty et al. found no significant matches with other genes. Whilst aligning the class I HLA genes in an analysis of HLA-J (10) we encountered this unusual sequence, which extends from positions 3826-4316 in the 3’ untranslated region of the HLA-F gene. In addition this sequence contains two poly A addition signals 90 bp downstream from the start of the 3’UTR (Fig. 1). When this entire sequence was used to search for sequence homologies, none was found. However, when the sequence was divided a significant homology between nucleotides 3997-43 16 and proteins of the large ribosomal subunit from a number of species was observed. This came from searching the Genbank and EMBL data bases, with the fragment 395 1-4316, using the Fasta program (provided by the GCG package) (1 1) (Fig. 2). Highest homology
3uTR Of HLA-F
-+
-C RiboMul prcin orientation
C I p u l O l i ~ ~
FulyA
FulyA
& j Ri~pocein-wrc
3792
3826 13597
I I I I
I I
3951
I
Segmentsuhidmwb
4316
Figure 1. Schematic representation of the 3’ untranslated region of HLA-F. Numbers indicate nucleotide positions in the sequence. Boxes show the region of the sequence that is homologous to other class I HLA genes or to the ribosomal proteins.
Brief Communication
50 1 MAPSAKATAA KKAVVKGTNG KKALKVRTSA TFRLPKTLKL ARRPKYASKA YSc L 2 5 MAPSTKAASA KKAVVKGSNG SKALKVRTST TFRLPKTLKL TRAPKYARKA YCu L 2 5 Mva L 2 3 Hma L 2 3 HLA-F Wheat L 2 3 Rice L 2 3 Maize L 2 3 Mpo L23 E u g r L23 E . C o l i L23 Y p s L23 Mca L 2 3 B s t L23
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
YSc L25 YCu
Mva L 2 3
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
:...... EE&
.--.:&:: ....
Hma L 2 3 HLA-F Wheat L 2 3 M R i c e L23 M Maize L 2 3 M Mpo L23 M Eugr L 2 3 M F Y F I S R K F Y E . C o l l L 2 3 ..MIREERLL Y p s L23 .MIREERLL Mca L 2 3 MHIT Bst L 2 3 .M@R
.........
......... ......... .........
.
...... ....
101
Hma L 2 3
............. ............. ............ ............ ...........
Wheat L 2 3
RIGPILGHTM H
Maize L 2 3
RMGPILGHTM KIGTTTGYTV RSNNFEGMKK
LFF L 2 3 LE'F E u g r L23 TAF E.C.011 L 2 3 KLF
MpO
150
.PGYSIPLLD
RETN.
RLGKWGKXP Figure 2. Alignment of the ribosomal protein-related sequences with the 3' ribosomal-like protein sequence of HLA-F. Boxed residues indicate identity between HLA-Fand the considered sequence. Sequences are from yeast: YSC, Succharomyces cnrlsbergensis (12)- YCU,Candidu utilis (13); Archuebacteria: Mvo, Methanococcus vannieli (14), Hma, Halobacteriwn mnrismortui (15); Plants: What, Triticwn aestivum (16), Rice, Orpa sutiva (17), Maize, Zea mays (18). Mpo. Marchuntia polyrnorpha (Liverwort) (19). Eugr, euglena gracilis (20);Eubacteria: E . Coli, Escherichiu coli ( 2 I), Yps, Yersinia pseudotuberculosis (22), M a . Mycoplrrsma capricolum (231, Bst, Bacillus stearothermophilur (24).
was found with the L25 protein of the yeast candida utilis, for which there is 60.5% sequence identity in the 329 bp region encompassing nucleotides
3997-4316. This results in 73% similarity or 54% identity of their deduced protein sequences. There are also significant similarities with the L23 protein 25 1
Zemmour & Parham
from bacteria and plants. Interestingly, the sequence from the HLA-F gene is more closely related to the ribosomal protein of yeast and archaebacteria than to those of plants and eubacteria (Fig. 3). Homologues of these ribosomal proteins in higher eukaryotes have not been previously described. The orientation of the reading frame for the ribosomal protein-like sequence is reversed from that of HLA-F (Fig. 1) and the sequence corresponds to the carboxy-terminal 87 residues of the 142 residue of the L25 protein. The sequence of HLA-F that is 3' to this region has yet to be sequenced, and thus it is not known,whether a sequence homologous to the entire L25 gene is represented in HLA-F. There is no evidence as to whether the ribosomal protein-like sequence that has been inserted into HLA-F is a processed or non-processed gene because the yeast L25 gene has no intronic sequence in the region homologous to HLA-F. Furthermore, it is possible that the unknown segment joining the HLA sequence to the ribosomal protein-like sequence (Fig. 1) could contain regulatory signals that may regulate the gene expression of the ribosomal protein. This junctional sequence contains two
c
Candida ufilu
Melhanocaccur vanniclii
Halobacnn~umman'smorlui --
poly A sites, one of which could belong to the ribosomal protein gene. The similarity of the 3' untranslated region of HLA-F with prokaryotic sequences raised the possibility that the presence of the ribosomal protein-like sequence could be an artefact of cloning. This was ruled out by the following experiment. A 3' oligonucleotide primer derived from the ribosome-related sequence was combined with a 5' primer derived from the class I-related sequence to amplify genomic DNA from different human Bcell lines. The polymerase chain reaction was performed in a volume of 100 pl, using 10 p1 of genomic DNA and 90 pmoles of each primer; HLA-F5p frGT GAC TGA TAC GAA TTT GTT CAT GI, HLA-F3p WC GAC TGG CTC CTG ACT ACG ATG C]. The amplification procedure consisted of 35 cycles involving a denaturation step of 1 min at 94"C, an annealing step of 1 min at 67°C and an extension step of 1 min at 72°C. Amplification products were then analyzed by gel electrophoresis on a 2% agarose gel. From the sequence of the cloned HLA-F gene this amplification was predicted to give a 365 bp fragment. Such a fragment was indeed the major product seen in the amplification from all 6 cell lines, showing that the ribosomal protein-like sequence present in the cloned HLA-F gene is not the result of a cloning artefact (Fig. 4). As a control the HLA-FSp primer was used in conjunction with the HLA-3pC primer that we have used for amplification of HLA-A,B,C cDNAs (25). In this case none of the 365 bp fragment was seen; instead, an expected fragment of 256 bp was obtained. As further controls, use of "a single oligonucleotide primer in the polymerase chain reaction with either HLA-F5p or HLA-F3p did not lead to DNA amplification. Using a probe derived from the 3' untranslated
Oryu ruriva
1 2 3 4 5 6 7 8 9101112
L
-
E $ c h e W h Coli
-
-
252
Eaglenu graeilia
Ycninia prerrrdolubrrcrlosu
M J C O P ~ eapricolurn M
Figure 4. PCR amplification of genomic DNA derived from the B cell lines; JY, T7527, LB, LBF, JHAF, IDF, (lanes 1 to 6 respectively) using HLA-FSp and HLA-F3p primers. Lanes 7-8 show amplified products obtained from the JY (lane 7) and T7527 (lane 8) using the HLA-F5p and HLA-3pC primers. PCR amplification using a single primer, either HLA-F5p (JY: lane 9; T7.527: lane 10) or HLA-F3p (JY: lane 1I, T7527: lane 12).
Brief Communication region of HLA-F for Southern blot analysis, Geraghty et al. found 30-50 bands under conditions of low stringency compared to the single HLA-F band revealed under conditions of high stringency (6). That probe is now known to include the ribosomal protein-like sequence. This indicates, first, that there are many sequences related to the yeast L2.5 ribosomal protein in the human genome and, second, that the sequence found in the HLA-F gene is likely to be a unique variant of this sequence. From northern blot analysis, Geraghty et al. showed that some of these sequences were expressed in many tissues, suggestingthat they corresponded to “housekeeping genes” of which those encoding ribosomal proteins would be classic examples. A transcript of 600 bp was particularly prominent and is of sufficient size to encode a ribosomal protein like L25. However, the functional significance of the ribosomal-like sequence in the 3‘ untranslated region of HLA-F, and whether it is transcribed independently of the class I-like sequences of this gene, are questions that have yet to be answered. Acknowledgments
This research was supported by a Shannon Award (A1 242.58) from the NIH. References I . Heinrichs H, Orr HT. HLA non-A,B,C class I genes: their structure and expression. Immunol Res 1990: 9: 265-74. 2. Shimizu Y, Geraghty DE, Koller BH, Orr HT. DeMars R. Transfer and expression of three cloned human non-HLAA,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc Natl Acad Sci USA 1988: 85: 227-31. 3. Ellis SA, Palmer MS, McMichael AJ. Human trophoblasts and the choriocarcinorna cell line BeWo express a truncated HLA class I molecule. J Imrnunol 1990: 144: 731-5. 4. Kovats S, Main EK, Librach C, Stubblebire M, Fisher SJ, DeMan R. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990: 248: 220-3. 5. Wei X, Orr HT. Differential expression of HLA-E, HLAF, and HLA-G transcripts in human tissue. Hwn Immunol 1990: 29: 131-42. 6. Geraghty DE, Wei X, Orr HT, Koller BH. Human leukocyte antigen F (HLA-F). An expressed HLA gene composed of a class I coding sequence linked to a novel transcribed repetitive element. J Exp Med 1990: 171: 1-18. 7. Lury D, Epstein H, Holmes N. The human class I MHC gene HLA-F is expressed in lymphocytes. Int Immunul1990: 2: 531-7. 8. Vemet C, Chimini G, Boretto J, Le Bouteiller P,Pontarotti P. Organization and Evolution of the MHC Chromosomal Region: An Overview. In: Klein J, Klein D. eds. Molecular Evolution of the Major Histocompatibility Complex, Berlin, Heidelberg: Springer-Verlag, 1991: 1-1 1. 9. Lawlor DA, Ward FE. Ennis PD, Jackson AP, Parham P. HLA-A and B polymorphism predate the divergence of humans and chimpanzees. Nature 1988: 335: 268-71.
10. Messer G, Zemmour J, Orr HT, Parham P, Weiss EH,
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