~ HMG-1
Nucleic Acids Research, Vol. 19, No. 23 6643
,..) 1991 Oxford University Press
Sequence of human HMG2 cDNA
A.Majumdar, D.Brown, S.Kerby, I.Rudzinski, T.Polte, Z.Randhawa and M.M.Seidman* Otsuka Pharmaceuticals, 9900 Medical Center Drive, Rockville, MD 20850, USA EMBL accession no. X62534
Submitted October 18, 1991 We have cloned a cDNA from human cells which encodes the human HMG-2 gene (1). During the course of isolation of a growth factor from medium conditioned by phorbol ester treated monocytic leukemia cells (U937) we isolated an additional protein with the N terminal sequence of GKGDPNKPRGKM. A cDNA library was prepared from mRNA isolated from phorbol ester treated U937 cells and clones identified by hybridization with an oligonucleotide probe based on the protein sequence. The sequence analysis of the longest insert revealed a cDNA of 1288 nucleotides containing a 215 nucleotide 5' untranslated region, an open reading frame of 627 nucleotides, and a 446 nucleotide 3' untranslated region. The sequence of the translated protein shows a very high degree similarity to the published porcine HMG2 protein (2) (Figure 1). There are only two differences in the human sequence; an S in place of a G at residue no. 168, and the deletion of one E in the run of E in the acidic C terminal region of the protein (Figure 1). The cDNA sequence shows an overall 79 % identity with the porcine sequence. The 5' untranslated region has a reduced GC content (58%) relative to the pig cDNA while the 3' untranslated region retains the high AT content of the pig (74% vs 71 %). Comparison of the HMG2 protein with human HMG1 protein indicates that the HMG2 sequence contains two HMG domains and shows 86% identity with the HMG1 in these domains. These domains are found in other DNA binding proteins and transcription factors (3, 4). In the acidic C terminal region there is only a 50% identity with HMG1 across the HMG2 sequence. The cDNA insert was used as a probe to analyze mRNA size and expression in U937 cells following phorbol ester treatment. A single band of approximately 1300 nucleotides was found suggesting that the clone was likely full length (Figure 2). There was a 3-fold decline in level after 48 hours of treatment.
*
To whom correspondence should be addressed
ACKNOWLEDGEMENTS
We thank Drs Michael Bustin and David Landsman for helpful advice and encouragement.
REFERENCES
1. Bustin,M., Lehn,D.A. and Landsman,D. (1990) Biochim. Biophys. Acta 1049, 231-243. 2. Shirakawa,H., Tsuda,K. and Yoshida,M. (1990) Biochemistry 29, 4419-4423. 3. Jantzen,H., Admon,A., BeHl,S.P. and Tijan,R. (1990) Nature 344, 830-836. 4. Kolodrubetz,D. (1990) Nucl. Acids Res. 18, 5565. domain
human HMG-2 pi g HMG-2 human HMG-1
MGKGDPNKPRGKMSSYAFFVQTCREEB00HI5PDSSVNFAEFSKKCSERWKTMSAKEKSKFEDMAOSDKARYDREM
human HMG-2 pig HMG-2
KNYVPPKGDKKGKKKDSPMKRPPSAFFLFCSEHRPKIKSEBPGLSIGDTAKKLGEMWSEQSAKDKQPYEQKAAO
human HMG-1 human HMG-2
pig HMG-2
human HMG- 1 215
.... .... ............................................ . 5 K. ...... K.... .E..
...
75 75 75
........................................................................... V. T.I .... ET.K............... Y. G. NNTA.D . K....
150 150 150
LKEKYEKDIAAYRAKGKSEAGKKGPGRPTGSKKKNEPEDEEEEEEEE-DEDEEEEDEDEE
209
K...................GE
. ................
A. ..VVI( K .........PD
Y
210
KED. ED. ... D D.EE ED........ EDDDDE
Figure 1. Sequence similarities between human HMG2 and porcine HMG2, and human HMG1. Non identical residues are indicated, as are the location of the two HMG1 domains.
Figure 2. Northern blot analyses of poly A+ RNA from U937 cells treated with phorbol ester for times as indicated. Analyses of the blot with a probe for GPDH indicated that equivalent amounts of RNA were loaded in all lanes.
Nucleic Acids Research, Vol. 19, No. 23 6643
,..) 1991 Oxford University Press
Sequence of human HMG2 cDNA
A.Majumdar, D.Brown, S.Kerby, I.Rudzinski, T.Polte, Z.Randhawa and M.M.Seidman* Otsuka Pharmaceuticals, 9900 Medical Center Drive, Rockville, MD 20850, USA EMBL accession no. X62534
Submitted October 18, 1991 We have cloned a cDNA from human cells which encodes the human HMG-2 gene (1). During the course of isolation of a growth factor from medium conditioned by phorbol ester treated monocytic leukemia cells (U937) we isolated an additional protein with the N terminal sequence of GKGDPNKPRGKM. A cDNA library was prepared from mRNA isolated from phorbol ester treated U937 cells and clones identified by hybridization with an oligonucleotide probe based on the protein sequence. The sequence analysis of the longest insert revealed a cDNA of 1288 nucleotides containing a 215 nucleotide 5' untranslated region, an open reading frame of 627 nucleotides, and a 446 nucleotide 3' untranslated region. The sequence of the translated protein shows a very high degree similarity to the published porcine HMG2 protein (2) (Figure 1). There are only two differences in the human sequence; an S in place of a G at residue no. 168, and the deletion of one E in the run of E in the acidic C terminal region of the protein (Figure 1). The cDNA sequence shows an overall 79 % identity with the porcine sequence. The 5' untranslated region has a reduced GC content (58%) relative to the pig cDNA while the 3' untranslated region retains the high AT content of the pig (74% vs 71 %). Comparison of the HMG2 protein with human HMG1 protein indicates that the HMG2 sequence contains two HMG domains and shows 86% identity with the HMG1 in these domains. These domains are found in other DNA binding proteins and transcription factors (3, 4). In the acidic C terminal region there is only a 50% identity with HMG1 across the HMG2 sequence. The cDNA insert was used as a probe to analyze mRNA size and expression in U937 cells following phorbol ester treatment. A single band of approximately 1300 nucleotides was found suggesting that the clone was likely full length (Figure 2). There was a 3-fold decline in level after 48 hours of treatment.
*
To whom correspondence should be addressed
ACKNOWLEDGEMENTS
We thank Drs Michael Bustin and David Landsman for helpful advice and encouragement.
REFERENCES
1. Bustin,M., Lehn,D.A. and Landsman,D. (1990) Biochim. Biophys. Acta 1049, 231-243. 2. Shirakawa,H., Tsuda,K. and Yoshida,M. (1990) Biochemistry 29, 4419-4423. 3. Jantzen,H., Admon,A., BeHl,S.P. and Tijan,R. (1990) Nature 344, 830-836. 4. Kolodrubetz,D. (1990) Nucl. Acids Res. 18, 5565. domain
human HMG-2 pi g HMG-2 human HMG-1
MGKGDPNKPRGKMSSYAFFVQTCREEB00HI5PDSSVNFAEFSKKCSERWKTMSAKEKSKFEDMAOSDKARYDREM
human HMG-2 pig HMG-2
KNYVPPKGDKKGKKKDSPMKRPPSAFFLFCSEHRPKIKSEBPGLSIGDTAKKLGEMWSEQSAKDKQPYEQKAAO
human HMG-1 human HMG-2
pig HMG-2
human HMG- 1 215
.... .... ............................................ . 5 K. ...... K.... .E..
...
75 75 75
........................................................................... V. T.I .... ET.K............... Y. G. NNTA.D . K....
150 150 150
LKEKYEKDIAAYRAKGKSEAGKKGPGRPTGSKKKNEPEDEEEEEEEE-DEDEEEEDEDEE
209
K...................GE
. ................
A. ..VVI( K .........PD
Y
210
KED. ED. ... D D.EE ED........ EDDDDE
Figure 1. Sequence similarities between human HMG2 and porcine HMG2, and human HMG1. Non identical residues are indicated, as are the location of the two HMG1 domains.
Figure 2. Northern blot analyses of poly A+ RNA from U937 cells treated with phorbol ester for times as indicated. Analyses of the blot with a probe for GPDH indicated that equivalent amounts of RNA were loaded in all lanes.
