isolation of a cdna encoding the chicken p50b/p97 (lyt-10) transcription factor
TRANSCRIPT
Gene, 138(1994)193-196 0 1994 Elsevier Science B.V. All rights reserved. 0378-1119/94/$07.00
SSDI 0378-l 119 (93) E0577-Z
193
GENE 0758 1
Isolation of a cDNA encoding the chicken p50B/p97 (Lyt-10) transcription factor
(HIV; lymphoma; h library; c-Rel oncoprotein; NF-KB; nuclear localization; ankyrin repeat domain)
Toshio Ikeda”, Yoshikazu Hirotaa and Takashi Onoderab
aNational Institute of Animal Health, Tsukuba, lbaraki 305, Japan. Tel. (81-298 ) 38-7842; and bFaculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyoll3, Japan.Tel.(81-3)3812-2111
Received by T. Sekiya: 30 June 1993; Revised/Accepted: 10 August/l2 August 1993; Received at publishers: 6 September 1993
SUMMARY
NF-KB is a transcription factor composed of the ~50 and p65 subunits. Recent works identified another human gene
which encodes a molecule related to the ~50 subunit, termed p50Z3, ~49 or lyt-10. Here, we isolated the cDNA clones
encoding chicken p50B/p97 (Lyt-10). The deduced amino acid sequence of the precursor protein, ~97, shows conservation
of the overall structure, and 86% identity in the Rel homology domain (RHD) and 56% identity in the ankyrin repeat
domain (ARD) to human p50B/p97. Expression of this gene is highest in the chicken spleen.
INTRODUCTION
NF-KB is a transcription factor and composed of a
heterodimer of ~50 and ~65 subunits (Baeuerle and
Baltimore, 1988). Both subunits contain the RHD at their
N-terminal portion which is responsible for DNA bind-
ing, dimer formation and association to some proteins
containing the ankyrin repeats (Liou et al., 1993). The
~50 subunit is first synthesized as a precursor protein,
~105, and matured through elimination of the C-terminal
ankyrin repeats domain (ARD) by proteolysis. Recently,
three groups independently isolated another gene related
Correspondence to: Dr. T. Ikeda, at his present address: Institute for
Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606,
Japan. Tel. (81-75) 751-3990; Fax (81-75) 751-3991; e-mail:
Abbreviations: aa, amino acid(s); ARD, ankyrin repeat domain; bp, base
pair(s); HIV, human immunodeficiency virus; kb, kilobase or 1000 bp;
Lyt-10, human p50B/p97; NF-h-B, nuclear transcription factor rB; nt,
nucleotide(s); ORF, open reading frame; pSOB, transcription factor re-
lated to the NF-kB/Rel family; p50B/p97, gene encoding p50B/p97; ~97,
precursor protein of p50B; PCR, polymerase chain reaction; Rel, onco-
protein and transcription factor coded by rel; RHD, Rel homology
domain.
to the ~50 subunit, and termed as p49/plOO, lyt-10 or
p50B/p97, respectively (Neri et al., 1991; Schmid et al.,
1991; Bours et al., 1992). Here, we use the term pSOB/p97
for this gene. p97 represents the precursor form of p50B.
This gene is mapped to the human chlOq24 that is the
chromosomal breakpoint of some T cell acute lympho-
blastic leukemia and B cell lymphoma. p50B stimulate
the transcription dependent on the HIV KB site in syn-
ergy with ~65 and RelB (Schmid et al., 1991; Bours et al.,
1992). More recently, it has been shown that the homo-
dimer of p50B associates with the oncoprotein Bcl-3 and
directly transactivates the expression of HIV through the
HIV KB motif (Bours et al., 1993). p50B, therefore, is
thought to be involved in the regulation of cellular and
viral gene expression.
EXPERIMENTAL AND DISCUSSION
(a) Isolation of a cDNA encoding chicken p50B/p97
(Lyt-10)
We have cloned the cDNAs encoding chicken
p50B/p97 from chicken spleen cDNA library in hgtll
and determined the nt sequence of one of them containing
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A CCCGCCCTCGAGGTCGACGATGGCTGTGCGGCACACCAGGTGTGAGTAGGAG~TGGAGCGGGACCTCCAGGCGG~GTAGGACTCCATCATGTGCCGCACCTTCTCCGTCACGTTGTA GTAGAGGTGGGCACTCTGCAGGGGGACCTTGCCCTCCTGGCCCAGCTTGAGGGCCTTCAGGAGGTGACACCGTAG~GGTCTCCGCGAGGGGCCGGGGGAGAGCGGCGGTCCGGAGGAGA AGGGATGTGGAGGAAGCGATCTCACCCCGCGCTGCCCTGACCCTGCCCTCCGCCCG~CTCCGTGACGATTCCCTGCGGC~CCCTGACCCGGCGGTGCCCGCGGCGATGCTGGGGCTG GACGGGCTGCTGCGGCCGGCCGCCTCCGGCACGGCCGGCGGCCGGCCCCGCGGCGACATGGACGAGCACTTCCAGCCCTGCCTGGATGGGATTGACTACGATGACTTCAGCTTCGGCTCG
MDEHFQPCLDGIDYDDFSFGS CACATGGTGGAGCAGAAGGAGCCCCTGATGGAGACAGCAGTCGGGCCCTACCTGGTCATCATCGAGCAGCCG~GCAGCGGGGCTTCCGATTTCGGTATGGCTGCGAGGGCCCTTCTCAC HMVEQKEP LMETAVGPYLVIIEQ P K Q RGFRFRYGCEGPSH GGGGGGCTGCCAGGCGCCTCCAGCGAGAAGGGGGCAC~GACCTATCCCACCGTC~GATCTGC~CTACGAGGGGATGGCGCGCATCGAGGTGGACCTGGTGACGCACAGCGACCCTCCG GGLPGAS SEKGHKTYPTVKICNYEGMARIEVDLVTHSDPP CGTGTGCACGCGCACAGCCTGGTGGGCAAGCRGTGCAACGCTGGGTGTGCTCCACGTCACC RVHAHSLVGKQCNEAGNC VAIVGPKDMTAQFSNLGVLHVT llAGAAGAACATGATGGAGATCATGAAGGAGAG~GCTG~G~GCAG~GACGCGC~CAC~TGGGCTGCTGACAG~GCTGAGCTGCGTGAGATCGAGCTGGAGGCC~GGAGCTG~G KKNMMEIMKEKLKKQ KTRNTNGLLTEAELREI ELEAKELK AAGGTGATGGACCTGAGCATCGTGCGGCTGCGCTTCACCGCTTACCTCCGTGACAGCAGTGGG~CTTCACTCTGGCACTACAGCCCGTCATCTCTGACCCCATCCATGACAGC~GTCC KVMDLS IVRLRFTAYLRDSSGNFTLALQPVISDPIHDSKS CCCGGCGCTTCCAACCTGAAGATCTCGCGGATGGAC~GACTGCGGGCTCAGTGCGGGGTGGGGACGAGGTGTACCTGCTGTGCGAC~GGTGCAG~GATGACATTGAGGTGCGGTTC PGASNLKISRMDKTAGSVRGGDEVYLLCDKVQKDDIEVRF TATGAGGACGACGAGAACGGCTGGCAGGCCTTCGGGGACTTCTCCCCCACGGACGTACAC~GCAGTACGCCATCGTCTTCCGCACGCCCCCCTACCAC~GCCC-TTGACCGTCCT YEDDENGWQAFGDFS PTDVHKQYA IVFRTPPYHKPKIDRP GTCACCGTGTTCCTGCAACTGAAGCGGAAGCGG~GCGCGGTGGGGACGTCAGCGACTCC~GCAGTTCACCTATTACCCTGTGGTGGAGGAT~GGAGGAGGTGGAGCGG~GCGC~G~GGTG VTVFLQLKRKRGGDVSDSKQFTYYPVVEDKEEVEIRK R K KIV CTGCCTCAGTTTCCCCAGCACTTCGGCGGGGGCTCACACATGGGGGGTGCCGGCGGTGCTGGGGGCTTTGGGGCAGGAGGAGGCGGT~CCTCAGCTTTCCTTACTCATCTGGACTGGGC LPQFPQH FGGGSHMGGAGGAGGFGAGGGGNLSFPYSSGLG TACAACAACCTCTACTCCTCCAGCCCGCACCCTGTGGGGGGTGGGTACCAGGGCGGCGTGCAGATG~GGCTGCCAGCGAGAGCGGGGATGGAGATGACAGACAGGCGCCCACAG~GT YNNLYSS SPHPVGGGYQGGVQMKAAS ESGDGDDRQAPTES ACCTATTGCAGGGAGCTGCAGCGGCACGCCCACTTGTGCCACCTGTGGCTGCTGGCACGCCGC~CGCCCATGCCCTGCTGGACTACTCGGTGACTGCTGACCCCCGCATGCTGCTGGCC TYCRELQ RHAHLCHLWLLARRNAHALLDYSVTADPRMLLA GTGCAGAGGCACCTGGCTGCCTCGCAGGATGAGAATGGGGGACACGCCCTTGCACCTCGCCATCATCCATGAGCAGACGGCTGTGATC~GCAGCT~TTGAGGTGGTGGTCAGCATCCCT VQRHLAASQDENGDTPLHLAII HEQTAVIKQL IEVVVSIP AGCCAGCAGATCATTAACATCACCAACRACTTGCAGCAGACGCCACTGCACCTGGCGGTCATCACC~GCAGCCCCAGGTGGTGCAGCTCCTGCTGCAGGCCCACGCC~CCCCACCCTG S Q Q 1 INITNNLQQT PLHLAVITKQ PQVVQL L L Q A HANPTL CTGGACCGCTACGGCAACTCCCTGCTGCACCTGGCACTGCAGGCGGCTGATGAGGAGATGCTGCGGATGCTGCTGGCCCACCTGGGCTCGGCCACTCCCTACCTGCTGCACCTGCCC~C LDRYGNSLLHLALQAA DEEMLRMLLAHLGSATPYLLHLPN TTCCAGGGTCTCCTGCCCGTACACCTGGCTGTG~GGCG~GAGCCCGGCCTGCTTGGACCTGCTGGTCAGG~GGGTGCGGATGTG~CGGCGTGGAGAGGCAGGGTGGCAGGACCCCG FOGLLPVHLAVKAKS PACLDLLVRKGADVNGVERQGGRTP CTGSACCTGGCCGTGGAGATGGAGAACCTCAACATGGCCACGCACCTGGTG~G~GCTGGGAGC~TGTC~CAGCCGGACCTTTGCCGGG~CGCCCCC~TGCACCTGGCTGCCGGC LHLAVEMENLNMATHLVKKLGANVNSRTFAGNAPLHLAAG CTGGGCTCCCCAACCCTCACCAAACTGCTGCTGCTT~GCAG~GCAGATGTGCAGCGTGAG~CGATGAGCCCGTCAGCCCCTCCTCGTCAGAGGCCAGCAGCGACACGGATGGCGACCCC L G S PTLTKLLLKAGADVQRENDE P v s PSSSEASSDTDGDP GAGGAGCAGGAGCAGGAGCAGGCCATGGAGCTGGGAGAGCCGGCTCTGAGCCCCCATCCCACCCCCGAGGAGGAGCAGGAGGA~CGGGGCCCCGGCAGCGCCGCTGCCACACAGCCCTG EEQEQEQAMELGEPALS PHPTPEEEQ EEAGPRQRRCHTAL GACCTGACCCGGAGCCAGAGTGCGGGACATCCTGCTGCAGGCCTCCCAGCCGCGCCCCGATACTGAGCTGCCCACCACCCCCCGGGCAGGG~CGTGCTGTCTCTGGACAGCGATGCG DLTRSQKVRDI LLQASQPR PDTELPTTPRAGNVLSLDSDA CTGCAGGGGCTGGAGCAGCTGCTGAATCAGGACGGCAGGACGGCAGCGGGTCGGACTGGATGGAGCTGGCC~GAGGCTGGGGCTCTGCAGCCTTGTGGAGACCTAC~GGACACCCCTTCGCCCAGC L Q G L E Q L LNQDGSGSDWMELAKRLGLCS LVETYKDTPSPS GTCAGCCTGCTGCGCAGCTACGAGCTGGCCGGGGGCAGCCTTGGGGGGCTGCTGGAGGCTCTGGACTCCATGGGGCTGCGCGGAGCTGTCAG~TGCTGCGC~CCCGAGCCGCTGGAG VSLLRSYELAGGSLGGLLEALDSMGLRGAV RMLRKPEPLE ARGCTGCAGAGCACAGAGGTCARGGAAGACAGTGCCTATGGGAGCGAGTCGGTGGAGGAGGAGCAGGCGGCCGCCCTG~GCCGAGGCCGGTGCCAGAGGGCGAGCTGCCCCACAGCCAG KLQSTEVKEDSAYGSESVEEEQAAA LKPRPVPEGELPHSQ CAGCAGCAGCAGGTGCACTGAGGGGCCGGGGGGGCCGCGCCGCCCCCCGCCCCGCCCCTCCCACAC~GTGCCTTCTGGACTGGGGGCTGCGCTGCAGCCCGGCTGCCCTGCTGCTGAGG QQQQVH* GCCGGGACACGGCGCGCCCGGCCCCGCTCTGCTCTTATTT~CGGTCCCGCGCCTGGTG~T-GGGGACACGGCTTCCCTTAGCCTC~
B
401 450 Chicken G@YQ&@@K &AS@Z$@DD R@PTESTYC RELQRHAHL. .........C HUdLU$RIU@ HRfrL?XS~&
Human F@a@&@@ #FVI?$@SGE E&EPSAPSR TPQCDAQAPE MLQRAREYNA &F&.i@Sh F!&$&%~~
~Glycine Rich Region End Ankyrin Repeat8 Domain Start
120 240 360 480 21 600 61
720 101 840 141 960 181
1080 221
1200 261 1320 301
1440 341
1560 381
1680 421
1800 461
1920 501
2040 541
2160 581
2280 621
2400 661
2520 701 2640 741
2760 781
2880 821
3000 861
3120 901
3240 941
3345
901 927 V@E@&PaSQ QQQQ%?W
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the entire ORF and other clones in both strands. The
3345-bp sequence codes an ORF (from nt 418 to nt 3138;
940 aa long). The calculated M, of p97 is 99402, and is
slightly smaller than that of ~105 (Fig. 1A). The entire
structure of chicken p50B/p97 is composed of Rel homol-
ogy domain (RHD), glycine-rich domain, and ankyrin
repeats domain (ARD) from the N terminus to the C
terminus (Fig. 1B). This structure is similar to that of
human ~97, and mammalian and chicken ~105. The de-
duced aa sequences of RHD and ARD of chicken p97
show 86% and 56% identity with those of human ~97,
respectively. A nuclear localization signal (RKRKK) (aa
336-340, Fig. lA), which is conserved among Rel/NF-KB
related proteins, is also conserved at the C-terminal end
of RHD of chicken p50B/p97. However, there is no
putative phosphorylation site for protein kinase A
(RRXS) which is conserved in other Rel/NF-KB related
proteins. Therefore, post-translational regulation of
p50B/p97 could be different from other Rel/NF-KB re-
lated proteins.
(b) Expression of chicken p_W/B/p97 (lyi-IO) in various
organs
Northern blot analysis revealed that the expression of
this gene was highest in chicken spleen, and chicken B
cell tumor cell lines (Fig. 2). Its expression pattern is
similar to those of @O/p105 and c-rel (Ikeda et al., in
press). The human p50B/p97 gene is located to the chro-
mosomal breakpoint in some leukemia cells (Neri et al.,
1991). The candidate oncoprotein Bcl-3, which associates
with the homodimer of p50B, is also located to the chro-
mosomal breakpoints of some B cell leukemia cells (Ohno
et al., 1990) and transactivates through KB motifs (Bours
T B m-
1 2 3 4 5 6 7 8 9 i0 lli2
Fig. 2. Northern blot analysis of chicken pNBip97. Poly(A)-selected
RNAs (5 ug per lane) prepared from 8-week-old chickens (lanes l-7)
and chicken cell lines (lanes 8-12) were fractionated on a gel, and
hybridized with a probe specific to p50B/p97. BF indicates the bursa
of Fabricius. T and B indicate chicken T and B cell lines, respectively.
Migration of 28s and 18s ribosomal RNAs is indicated on the right.
Methods: Poly(A)+RNA was prepared from various organs of 8-week-
old chickens (White Leghorn) by the method described above. The
poly(A)+RNA (5 yg each) was electrophoresed on a 1.0% agarose gel
containing 2.2 M formaldehyde and then blotted to a Hybond N+
membrane (Amersham). The blots were hybridized to the following
probe labeled with [c(-32P]dCTP in the hybridization buffer
(5 x SSC/SO% formamide) at 37’C. The probe used was prepared from
a SalI-Sphl fragment corresponding to nt 13-1791 (Fig. IA) by mega-
prime kit (Amersham). The membranes were finally washed with
0.2 x SSC/l% SDS at 5O’C. Radioactivity of the filter was analyzed
with a Fuji Bio-Image analyzer BAS2000. SDS, sodium dodecyl sulfate:
SSC, 0.15 M NaCl,/O.OlS M Na,citrate pH 7.6.
et al., 1993). These observations suggest that these gene
products might play an important role(s) in controlling
the growth and differentiation of lymphoid cells.
Fig. 1. Sequences of p5OB/p97 (Lyt-IO). (A) The nt sequence of chicken pX)B/p97 (/yt-f0) cDNA and deduced aa sequence. The deduced aa sequence
starting from the putative start codon at nt 418-420 is indicated under the corresponding codons. The nt and aa residues are numbered at the right
end of every lane. The stop codon is marked with an asterisk. The nuclear localization signal sequence (RKRKK; aa 336-340) is boxed.
Poly(A)-addition signal sequence (nt 3301-3306) is underlined. The nt sequence data reported here will appear in the DDBJ. EMBL and GenBank
Nucleotide Sequence Databases with accession No. D16367. (B) Comparison of aa sequences of chicken and human p50B/p97 (Lyt-IO). The identical
aa residues between two aa sequences are shaded. Rel homology domain, Gly-rich region and ARD are indicated by arrows under the aa sequence.
Ankyrin repeats are underlined. Methods: (a) Preparation of the DNA probe for chicken p5OB/p97 (lyt-f(l): The fragment used for the library screening
was prepared by a reverse-transcription aided polymerase chain reaction (RT-PCR). Total RNA was extracted from an adult chicken spleen by the
guanidium thiocyanate/CsCl method (Chirgwin et al., 1977). A cDNA pool was generated using the total RNA, Mu-MLV reverse transcriptase (BRL)
and oligo(dT) as a primer. Oligo(dT)-primed cDNA of chicken spleen was used as template for PCR. Used primers are as follows: primer 1.
5’-TTTCGATATGGCTGTGAAGG and primer 2, S-TCRTCYTTYTGNACYTTRTC. PCR was performed using the GeneAmp kit (Perkin-Elmer-
Cetus) and a thermal cycler ( Perkin-Elmer-Cetus). Each cycle consists of incubation at 94’C for 1 min, followed by 1 min annealing at 50,-C and by
extension for 1 min at 72 ‘C. After 30 cycles, the extension at 72’C was prolonged to 10 min. The reaction mixture (50 ~1) included 5 ul of the reverse
transcribed cDNA/50 pmol of each primer/O.2 mM each dNTPj50 mM KC]/10 mM TrisHCl (pH 8.3)/1.5 mM MgCl,/O.Ol% gelatini2.5 units of Tag
polymerase. DNA fragments amplified in the PCR were fractionated by electrophoresis in 1.5% agarose gel, and the appropriate fragments were
cloned into the Sum1 site of pBluescript II, and sequenced by the dideoxy chain termination method (Sanger et al., 1977) with Sequenase Version 2.0.
(US Biochemicals, Cleveland, OH, USA). following the manufacturer’s instructions. (h) Construction and screening of a chickerrspleen cDNA library:
A cDNA library was constructed in hgtll vector (Stratagene, La Jolla, CA, USA) using oligo(dT)-selected chicken spleen poly(A)+RNA as a template
(Hyunh et al., 1985). Approximately IO6 recombinants were screened with the probe mentioned above. Phage clones positive in the plaque hybridization
were purified, and their inserts were subjected to sequencing after subcloning into pBluescript II. A series of deletion clones were prepared by ExoIII
and Mung Bean nucleases of the kilodeletion kit (Takara), then sequenced as described above. The primers used were a universal and a reverse
primer (Stratagene), and synthetic oligodeoxynucleotides generated according to newly identified internal sequences.
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(c) Conclusions
(I) cDNA encoding chicken p50B/p97 (Lyt-10) was
isolated. The deduced aa sequence of chicken p50B/p97
(Lyt-10) showed approximately 86% identity in the RHD
and 56% in the ARD to human p50B/p97 (Lyt-10).
(2) Chicken p50B/p97 (lyt-IO) mRNA (3.6 kb) was
highly expressed in the spleen and B cell lines.
ACKNOWLEDGEMENTS
We thank Dr. Matuda for his generous gift of the
chicken cell lines and Dr. S. Itohara for critical reading
of the manuscript and helpful discussion. We also thank
K. Yoshinari for technical assistance. This work was sup-
ported by the special coordination funds for promoting
science and technology from the Science and Technology
Agency of Japan.
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