GENOMICS

13, 665-671

(1992)

Characterization and Chromosomal Mapping of the Gene Encoding the Cellular DNA Binding Protein ILF CHING LI,*,’ ALDONS J. LUSIS,* ROBERT SPmKEs,t *Department of Medicine,

December

19, 1991;revised

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-l LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene. 0 1992 Press,

AND RICHARD GAYNOR*

of Medicine and Department of Microbiology and Molecular Genetics, and tDivision of Medical Genetics, Department University of California, Los Angeles, California 90024; and SDivision of Molecular Virology, Department of Medicine and Microbiology, University of Texas Southwestern Medical School, Dallas, Texas 86235 Received

Academic

AJAY NIRULA,+

Inc.

INTRODUCTION

Regulation of viral and cellular gene expression is dependent on the interaction of cellular transcription factors with upstream promoter elements (Mitchell and Tjian, 1987). Similar purine-rich regulatory elements are found in a variety of viral and cellular promoter elements, including those of the human immunodeficiency virus (HIV-l), human T-cell leukemia virus (HTLV-I), and interleukin-2 (IL2) gene (Durand et al., 1988; Crabtree, 1989; Bosselut et al., 1990; Gitlin et al., 1991). Several cellular factors have been demonstrated to bind to these motifs. A T-lymphocyte-inducible cellular factor, known as nuclear factor of activated T-cells (NFAT), binds to similar motifs in the HIV-l LTR and IL2 promoter (Durand et al., 1988; Crabtree, 1989). Members of the ets family of transcription factors also bind specifically to purine-rich sequences in both viral and cellular ’ Current address: Institute of Biomedical Science, National Hua University, Hsinchu 300, Taiwan, Republic of China.

Tsing

February

26, 1992

promoters (Karim et al., 1990; Klemsz et al., 1990; Gitlin et al., 1991). Recently we described the cloning of a cellular factor isolated from both HeLa and Jurkat cDNA libraries, known as ILF, that binds to purine-rich regulatory motifs in the HIV-l LTR and IL2 promoter (Li et al., 1991). The amino acid sequence of ILF contains several interesting amino acid homologies. These include sequences for potential ubiquitin-mediated degradation, a potential nucleotide binding site, and a nuclear localization signal (Li et al., 1991). The DNA binding domain of ILF contains strong homology to the Drosophila fork head gene (Weigel et al., 1989). Although the Drosophila fork head protein has not been demonstrated to bind DNA, members of a family of hepatocyte-specific transcription factors, known as HNF-3, have also been demonstrated to contain a very homologous DNA binding domain (Lai et al., 1990, 1991). HNF-3 regulates gene expression of several liver specific genes (Costa et al., 1989). However, the purine-rich sequences, to which ILF binds, are very different than the sequences bound by HNF-3 (Lai et al., 1990; Li et al., 1991). To further study the properties of ILF, we studied the binding of bacterial-produced fusion proteins containing either the full-length ILF protein or a portion of this protein containing only the fork head binding domain. We demonstrate that the ILF fork head domain alone is sufficient for binding to the same sequences as the entire ILF protein. ILF mRNA is alternatively spliced and is expressed constitutively in both lymphoid and nonlymphoid cells. The ILF gene is localized to chromosome 17q25, which is a site of rearrangement in some cases of acute myelogous leukemia. MATERIALS

AND

METHODS

Identification of different ILF cDNAs. A 706-bp portion of the ILF cDNA was identified using X gtll expression cloning with wildtype and mutated double-stranded ligated oligonucleotides, as previously described (Li et al., 1991). This fragment was labeled by random priming and used to screen a HeLa cDNA ZAP library (Stratagene) in an attempt to identify full-length cDNA clones. DNA sequence analysis of these clones employed the Sanger method sequence system. PCR

665

0888-7543/92

Copyright 0 1992 All rights of reproduction

by

$5.00

Academic Press, Inc. in any form reserved.

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analysis (30 cycles) with HeLa poly(A)RNA (10 pg) was performed to conclusively establish the existence of alternative splicing of ILF mRNA. HeLa poly(A)RNA (10 rg) was reverse transcribed using random hexamer primers and was used as a template for PCR (40 cycles: 94”C, 1 min; 72”C, 1 min; 55”C, 1 min). The following sets of oligonucleotides were used as PCR primers. Set 1: Sense (960) 5’-AGCTGATAGTTCAGGCGATT-3’ Antisense (2609) 5’-ACAGAGTTGATATCGTTAAA-3’ Set 2: (1713) 5’-AGGAGAATGGAGACCACAGGGAAGT-3’ (2019) 5’-CTGCTGTGTCAACTGAGGCA-3’

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Nucleotide positions correspond to the ILF-2 clone (Figs. l-3). GST-ILF fusion proteins. To prepare ILF in bacterial expression systems, ILF fragments were fused to the glutathione S-transferase gene (Smith and Johnson, 1988). A detailed discussion of the cloning strategy is given elsewhere (Li et al., 1991). A portion of the ILF extending from nucleotides 807 to 1620 (XmaI/SfiI) was cloned into pGEX-3X and gave rise to a 58kDa fusion protein. Another portion of the ILF cDNA extending from nucleotide 807 to 1075 (XmaI/ EcoRI) was inserted into pGEX-3X, and this fusion yielded a 37.kDa fusion protein. To construct a plasmid for bacterial expression of the ILF fork head DNA binding domain, a fragment extending from nucleotides 903 to 1252 in the ILF cDNA was first ligated to a Hind1111 Cl&digested pGEM-7Z vector (Promega). The pGEM-7Z vector containing the ILF fragment was digested with BumHI and SmuI for subcloning into the same sites of pGEX-3X. This fusion protein had a molecular weight of 40 kDa. All fusion proteins were induced by the addition of IPTG to Escherichiu coli containing the pGEX vectors and were purified by glutathione-agarose chromatography (Smith and Johnson, 1988; Li et al., 1991). Gel retardation analysis. Gel retardation was performed using an end-labeled 56bp AluI fragment extending from -310 to -255 in the HIV-l LTR or oligonucleotides corresponding either to an NFAT site in the HIV-1 LTR fragment extending from nucleotides -283 to -256 or to the NFAT site in the IL2 promoter between nucleotides -285 and -254. The sequences of these oligonucleotides are: HIV-1 LTR 5’-GAAGAGGCCAATGAAGGAGAGAACAACA-3’ IL2 51.AATTGGAGGAAAAACTGTTTCATACAGAAGGCGT-3’ Gel retardation was performed as described (Li et al., 1991) with 5 pg of each GST/ILF fusion protein. All incubation steps were performed at 4”C, followed by electrophoresis on 5% polyacrylamide gels with a low ionic buffer containing 6.7 mM Tris-HCl, pH 7.5, 3.3 mM sodium acetate, and 1 mM EDTA. E. co/i strains and preparation of bacterial expressed proteins. E. coli strains used were XL-1 Blue and TG-1. XL-l Blue was used for screening the HeLa X ZAP cDNA library, and TG-1 was used for the transformation of recombinant plasmids and the expression of the GST/ILF fusion proteins in bacteria. GST/ILF fusion proteins were prepared by induction of E. coli TG1 cultures with 0.5 mM IPTG at an OD,,, of 0.6-1.0 (Smith and Johnson, 1988). E. coli were harvested, lysed, and the supernatants were subject to glutathione-Sepharose 4B column chromatography (Pharmacia) for the purification of the fusion proteins as described by the manufacturer. DTT and PMSF were each added to all buffers to a 0.5 mM final concentration. Northern analysis. Cytoplasmic RNA (30 Kg) was obtained either from HeLa cells or from untreated, PMA (50 rig/ml) and PHA (1 pg/ml)-treated, or TNFL~ (500 U/ml)-treated Jurkat cells as described (Crabtree, 1989). RNA was analyzed by electrophoresis through 1% agarose formaldehyde gels, followed by Northern blot transfer to nitrocellulose as described (Gaynor et al., 1991). A 706-bp ILF EcoRI fragment comprising the original ILF binding domain and a 512-bp p-actin probe (Ponte et al., 1981) were labeled by random priming, hybridized to the filters, and subjected to autoradiography. Chromosomul mapping. A panel of 17 mouse-human somatic cell hybrid clones was constructed and analyzed for chromosome content as described previously (Mohandas et al., 1986). DNA was isolated from nuclei of these clones as well as from the parental mouse cell line (B82GM 0347A) and human lymphocytes using sodium dodecyl sul-

FIG. 1. Schematic of ILF cDNAs. A schematic of two ILF cDNAs isolated from a HeLa cDNA library is indicated. The coding sequences are represented by shaded areas. In ILF-1, the coding sequence extends from nucleotide 518 to 2147. The sequence for ILF-‘2 is identical with ILF-1 from nucleotides 518 to 1952 but differs by insertion of a 422-bp fragment at this point, which introduces a stop codon at position 2009. The region of homology with the Drosophilia fork head gene is indicated. fate (SDS) and proteinase K followed by phenol-choloroform extraction. Following cleavage with restriction enzymes, 10 pg of the DNA from each sample was electrophoresed through a 1.2% agarose gel and transferred by blotting to a nylon filter as described (Mehrabian et al., 1986). The filters were then probed with the ILF cDNA. After isolation by preparative gel electrophoresis, the insert was radiolabeled with [32P]dCTP by a random priming method (Feinberg and Vogelstein, 1983) to a specific activity of about 1 X 10’ cpm/mg (Mehrabian et al., 1986). Filter hybridization and autoradiography were performed as previously described (Mehrabian et al., 1986). For in situ hybridization, the ILF cDNA insert was labeled by random priming with “H-labeled deoxynucleotides to a specific activity of about 4 X 10’ cpm/mg. The probe was then hybridized to chromosomes from normal human lymphocytes using a method developed by Harper and Saunders (1981) as modified by Cannizzaro and Emanuel (1984). The slides were exposed for 10 days, and silver grains on or touching chromosomes were scored.

RESULTS Isolation of ILF cDNAs We previously reported the isolation of a cDNA which we designated ILF or interleukin enhancer binding protein using oligonucleotides corresponding to purine-rich regulatory elements to screen a X gtll expression library (Li et al., 1991). The fusion protein isolated from this cDNA bound to purine-rich regulatory sequences in the HIV-l LTR and IL2 promoter. DNA sequence analysis revealed that this protein known as ILF had strong homology in its protein coding sequence with the recently described fork head DNA binding domain (Weigel et al., 1989; Lai et al., 1990, 1991; Li et al., 1991). The ILF cDNA fragment was subsequently used to screen a HeLa cDNA library in an attempt to obtain full-length ILF cDNAs. Two cDNAs demonstrated in schematic form in Fig. 1 and designated ILF-1 (Fig. 2) and ILF-2 (Fig. 3) were isolated. However, it is likely that neither of these cDNAs is full-length.

CELLULAR

DNA

BINDING

PROTEIN

667

ILF 120 240 360 480

600 28 720 68 840 108 960 148 1080 188 1200 220 1320 266

TGGcGcccAGAcccmGAGGGAA

tz4GTTcGccGGccccc~Gc~Gc

GCTGYCATCCAGGMGCCC GGTmGcccAGAGcGc 1440 lZ%GCGCTGCACAGCCCAMCTC

CCCAGGCTCAC fi?lmmccAGTcAGcC

308

IQEARFAQSA

GAQTPESLSREGSPAPLEPEPGAAQPKLAV

cAcCccAGTGAccACmG4ccTcccAGcc

M~CCGTCCAGCG~~~~~~~CTA~~

PGSPLSSQPVLITVQRQLPQAIKPVTYTVATPVTTSTSQP

1560 348 1680 388 1800 428 1920 468

CCCcACTCCGGTccAcGGccA~

TGbXCGGCGAGTC ~~~~MMcAC~~C~~~CC~C~~~~

PTAVHGQVNNAAASPLHMLATHASASASLPTKRHNGDQPE

CGGTGAcuGaxGA

2040 508 2160 543 2280 2400 2520 2640 2760 2880 3000 3043

FIG. 2. Nucleotide sequence of the ILF-1 543-amino-acid open reading frame is shown. domain is shaded.

cDNA. The The portion

3043-bp nucleotide sequence of a partial ILF-1 cDNA and the portion of the open reading frame containing homology with the fork head

The sequence analysis revealed that both clones were identical except in their carboxyl-termini (Figs. l-3). Both cDNAs had an identical potential initiating methionine with a stop codon noted 138 bp upstream. In addition, both clones contained potential fork head DNA binding domains. However, in their carboxyl-termini, ILF-1 maintains an open reading frame until nucleotide 2147, while ILF-2 contains an additional 422 nucleotides inserted between positions 1953 and 2374, resulting in a protein that differs by 65 amino acids from the carboxyl terminus of ILF-1. To confirm the presence of these cDNAs, PCR analysis was performed using reverse transcribed HeLa RNA as a template. Using the primer sets described under Materials and Methods, we demonstrated that both cDNAs are expressed in HeLa cells. Primer set 1 flanks the putative alternatively spliced exon present in ILF-2 and not ILF-1. After 40 cycles of PCR with these primers a 1.2-kb product corresponding to ILF-1, but no product corresponding to ILF-2, was detected. This result could stem from a greater relative abundance of

containing DNA-binding

a

ILF-1 mRNA or from inefficient amplification of the larger ILF-2 PCR product (predicted size, 1.65 kb). Other smaller PCR products were also detected and their structures are currently being investigated. To confirm the presence of ILF-2 cDNA, we designed additional sets of primers with the antisense primer corresponding to sequences in the exon unique to ILF-2. Primer set 2 yielded the predicted PCR product size of 306 bp and was cloned and sequenced to confirm its identity as ILF-2. Appropriate negative controls were included for each PCR amplification. Thus at least two forms of ILF mRNA were present in HeLa cells encoding proteins of calculated molecular weights of 60 and 55 kDa, respectively. The functional significance of these two forms of ILF remains to be determined. The ILF Fork Head Domain Is Sufficient for DNA Binding A Drosophila gene known as fork head, which regulates terminal segment development, contains a llOamino-acid region that has a high degree of homology

668

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360 480 600 28 720 68 840 108 960 148 lo80 188 1200 228 ,320 268 14‘0 308 1560 348 1680 3.88 ,800 428 ,920 468 1040 497 2160 2280 2400 2520 2640 2160 2880 1000 3120 ,240 1160

FIG. 3. Nucleotide sequence of the ILF-2 cDNA. The 3465bp nucleotide sequence of a partial 497-amino-acid open reading frame is shown. The portions of the open reading frame containing domain and that region differing from ILF-7 are shaded.

with both ILF (Li et al., 1991) and a recently isolated hepatocyte-specific transcription factor HNF-3 (Lai et al., 1990, 1991), which regulates the hepatocyte-specific expression of the transthyretin and albumin genes (Costa et al., 1989). The sequences to which HNF-3 binds differ significantly from the sequences that ILF binds to in the HIV-l LTR and IL2 promoters. The sequences required for ILF binding also serve as binding sites for an inducible T-lymphocyte factor (NFAT); thus we refer to these regulatory motifs as NFAT binding sites (Durand et al., 1988; Crabtree, 1989). To determine whether the ILFforiz head domain alone was sufficient for binding to these NFAT motifs, we expressed different portions of the ILF gene as fusions with the glutathione S-transferase protein using the prokaryotic expression vector pGEX-3X (Smith and Johnson, 1988). These proteins were produced in E. coli and purified using glutathione-agarose affinity chromatography. Gel retardation was then performed using a fragment from the HIV-l LTR containing the distal

ILF-2 cDNA and the portion containing a homology with the fork head DNA binding

NFAT binding site (Fig. 4A), oligonucleotides corresponding to the HIV-l distal NFAT site (Fig. 4B), or the IL2 NFAT site (Fig. 4C). Competition analysis demonstrated the specificity of this binding (data not shown). Glutathione S-transferase protein alone did not bind to any of these probes (Figs. 4A, 4B, and 4C, lane 2). A fusion protein containing the majority of the ILF coding sequence bound specifically to each of these three probes (Figs. 4A, 4B, and 4C, lane 3). A truncated ILF protein containing primarily the fork head DNA binding domain also bound specifically to each of the probes (Figs. 4A, 4B, and 4C, lane 4). Surprisingly, the migration of the gel-retarded species using the complete ILF protein and the fork head domain alone were similar with the IL2 NFAT probe (Fig. 4C, lanes 3 and 4). Fusion proteins containing a deletion of most of the fork head domain did not bind to any of these probes (Figs. 4A, 4B, and 4C, lane 5). These results demonstrate that the fork head domain was sufficient to confer the binding specificity of the ILF protein to the NFAT binding sites.

CELLULAR

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DNA

BINDING

C

B HIV

HIV-oligo

I

I 1

2

3

4

5

669

ILF

PROTEIN

NFAT-oligo I

I 12345

FIG. 4. Gel retardation analysis of ILF. Gel retardation was performed with labeled DNA probes corresponding to either (A) an HIV-l LTR fragment extending from -310 to -265 (B) oligonucleotides corresponding to nucleotides -283 to -256 in the HIV-1 LTR or (C) oligonucleotides corresponding to nucleotides -285 to -254 in the IL-2 promoter. In A, B, and C, lane 1 contains no added extract, lane 2 contains glutathione-agarose-purified glutathione S-transferase (GST) alone, and lanes 3-5 contain similar purified GST/ILF-1 fusion proteins, with lane 3 containing a portion of the ILF protein extending from nucleotides 807 to 1620, lane 4 containing a portion of the ILF protein extending from nucleotides 903 to 1252, and lane 5 containing a portion of the ILF protein extending from nucleotide 807 to 1075.

ILF Is Constitutively

Expressed in a Variety of Tissues

IL2 gene expression is markedly increased upon the activation of T-lymphocytes due to an increase in the binding activity of specific cellular transcription factors (Crabtree, 1989). Recently, it was demonstrated that the cellular factor NFAT, which is critical for this activation of IL2 gene expression, is composed of a nuclear protein expressed in a wide variety of cells and a T-cell-specific cytoplasmic factor (Flanagan et al., 1991). Since ILF could potentially be related to the nuclear component of NFAT, we next determined whether ILF mRNA was constitutively expressed in epithelial and lymphoid cells. RNA was prepared from either HeLa cells or from a T-lymphocyte cell line, Jurkat, which was either unstimulated or stimulated with either PHA and PMA or TNF (Y. Northern analysis with an ILF probe revealed that two forms of ILF mRNA of approximately the same size were present in both HeLa (Fig. 5A, lanes 1 and 2) and Jurkat cells (Fig. 5A, lanes 2-4) and that the level of this RNA was not altered by activation of Jurkat cells. A longer exposure of this gel also revealed both mRNA species in the TNF a-treated Jurkat cells (data not shown). An actin probe revealed equal RNA loading in each sample (Fig. 5B, lanes l-4). In addition, we detected abundant levels of ILF in a variety of rat tissues, including kidney, heart, testes, lung, spleen, and intestines (data not shown). These results suggest that ILF is constitutively expressed in a variety of tissues. Chromosome Mapping of ILF The chromosomal localization of the ILF gene was determined by Southern blotting analysis of a panel of mouse-human genetic cell hybrids derived by fusion of

normal male fibroblasts (IMR 91) with thymidine kinase-deficient mouse B82 cells (Mohandas et al., 1986). The hybrids contained varying complements of human chromosomes as determined by karyotyping, and they have now been used for the chromosomal assignment of a large number of human genes. After digestion of genomic DNA prepared from the hybrid cell lines with HindIII, two major bands of 17 and 2.0 kb were noted upon blotting analysis with ILF cDNA (Fig. 6, lanes 1-14). Mouse genomic DNA alone yielded a hybridizing band of about 17 kb (Fig. 6, lane 16), while human DNA yielded only the 2.0-kb hybridizing band (Fig. 6, lane 17). B

A 12

34

1

2

3

4

4.0 kb -

FIG. 5. Northern analysis of ILF RNA. Northern analysis was performed with either a 706-bp ILF fragment, which was common to both ILF-1 and ILF-2 and extended from nucleotides 929 to 1634 in the ILF cDNA (A) or a 512.bp actin probe (B) using either 30 ~g of HeLa cytoplasmic RNA (lane 1) or similar amounts of RNA prepared from Jurkat cells untreated (lane 2), stimulated with PMA and PHA (lane 3), or stimulated with tumor necrosis factor (Y (lane 4). The position of the 4.0.kb ILF and 2.3-kb actin RNAs are indicated.

670

LI 1 2 3 4

5 6

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FIG. 6. Southern analysis of mouse-human somatic cell hybrids with ILF. DNA was isolated from a variety of previously described somatic cell hybrids including 84-24, 7, 20,21, 25,26,27, 30,34,35,37, 38, 39, and 116-5 (lanes l-15), a parental mouse cell line (lane 161, and a human cell line (lane 171, followed by digestion with HindIII. An ILF fragment was labeled by random priming and used in Southern analysis. The position of the 2.0.kb Hind111 fragment in human DNA is indicated.

Blotting analysis of HindIII-digested DNA from the panel of 15 mouse-human somatic cell hybrids revealed that the 2.0-kb human band cosegregated with chromosome 17, and all clones except 116-5 exhibited this band (Fig. 6, lane 15). Thus all the clones except 116-5 contained chromosome 17 (Fig. 6). Multiple discordancies were observed between the segregation pattern of the human ILF gene and all other chromosomes (data not shown). The regional localization of the ILF gene was examined by in situ hybridization to normal metaphase chromosomes. A significant accumulation of grains was observed only on the short arm of chromosome 17 with peak accumulation in the 17q25 region (Fig. 7). These results confirm the somatic cell hybrid analysis studies and further localize the ILF gene to human chromosome 17q25.

AL.

hancer. The binding domain of PU. 1 has homology with the DNA binding domain of ets oncogene (Karim et al., 1990; Klemsz et al., 1990). Finally, we have identified cellular factor ILF that binds specifically to NFAT sites in the HIV-l LTR and IL2 promoter (Li et al., 1991). Thus a number of factors are capable of binding to similar regulatory motifs. Two different ILF cDNAs were isolated. These cDNAs differ in their carboxyl-termini but otherwise have identical protein coding sequences. The significance of this difference remains to be determined. Both proteins possessa DNA binding domain that has strong homology with similar regions found in both the hepatic-specific transcription factor HNF-3A (Lai et al., 1990, 1991) and the Drosophila fork head protein (Weigel et al., 1989). We demonstrate that the ILF fork head binding domain is sufficient to allow for binding to HIV1 and IL2 NFAT motifs. The NFAT binding motifs have no apparent homology with the binding motifs of the hepatocyte-specific genes bound by HNF-3. Thus it is likely that the fork head binding domain can mediate binding to different regulatory sequences as seen with several other cellular transcription factors (Mitchell and Tjian, 1989). ILF mRNA is expressed in a variety of different cell lines. Since the purine-rich motifs, t,o which ILF binds, are found in the promoters of a number of cellular genes, it is possible that ILF is capable of regulating their expression. The localization of ILF to chromosome 17q25

12

l

21 1 21 2

a

21.3

22

a

23

DISCUSSION

A variety of cellular proteins that bind to similar purine-rich binding motifs in a number of viral and cellular enhancer elements have been described. One of these proteins known as NFAT binds to similar motifs in the IL2 and HIV-l enhancers upon stimulation of T-lymphocytes (Durand et al., 1988; Crabtree, 1989). Another cellular protein, a macrophage and B-cell-specific factor known as PU.1, binds to similar motifs in the SV40 en-

FIG. 7. Chromosomal localization of ILF by in situ hybridization. Metaphase chromosomes from normal human lymphocytes were hybridized with an ILF fragment labeled by random priming. The slides were exposed for 1 week and developed, and the grains touching chromosomes were scored. In an examination of 45 cells, the peak grain concentration occurred on chromosome 17, although a high background was noted. Of a total of 41 grains scored. 13 were on the long arm of chromosome 17 and 7 were on 17425.

CELLULAR

DNA

BINDING

is also of potential interest. This region is the localization site of genes for a homologue of the v-erb-A oncogene, procollagen, Z-oxoglutarate 4-dioxygenase, and protein disulfide isomerase (Solomon and Baker, 1989). In addition, the long arm of chromosome 17 is the site of translocations in human acute myelogenous leukemia. In particular, translocations of chromosome 11 and 17 t(11/17)(q23,q25) have been noted (Bloomfield and de la Chapelle, 1987). Interestingly a number of these translocations involve the displacement of ets-1 oncogene, which is located on llq23 (Watson et al., 1988). Since the Etsl protein can bind to NFAT motifs, it is interesting to speculate whether this translocation could potentially involve the substitution of the Etsl and ILF binding domains. Studies of clinical leukemia specimens will be required to determine whether the ILF is the site of potential translocations in human leukemia involved in this translocation. ACKNOWLEDGMENTS This work was supported by grants from the NIH (HL28481 and HL42438) to A.J.L. andby grants from the NIH, Veterans Administration, and the American Cancer Society to R.G. We are grateful for the technical assistance of Ivana Klisak, Ahn Diep, Yurong Xia, and Ping-Xi Wen.

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PROTEIN

close proximity to the Genet. 79: 352-356. Gaynor, during 2623.

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plasminogen.

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J. E., Jr. family in gene fork

Li, C., Lai, C., Sigman, D. S., and Gaynor, R. B. (1991). Cloning of a cellular factor, interleukin binding factor that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat. Proc. N&l. Acad. Sci. USA 88: 7739-7743. Mehrabian, M., Sparkes, R. S., Mohandas, T., Klisak, I. J., Schumaker, V. N., Heinzman, C., Zollman, S., Ma, Y., and Lusis, A. J. (1986). Human apolipoprotein B: Chromosomal mapping and DNA polynorphines of hepatic and intestinal species. Somt. Cell Mol. Genet. 12: 245-254. Mitchell, P. J., and Tjian, R. (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245: 371-378. Mohandas, T., Heinzmann, C., Sparkes, R. S., Wasmuth, J., Edwards, P., and Lusis, A. J. (1986). Assignment of human 3-hydroxy-3 methylglutaryl coenzyme A reductase gene to q13-q23 region of chromosome 5. Somt. Cell Mol. Genet. 12: 89-94. Ponte, P., Gunning, P., Blau, H., and Kedes, L. (1981). Human actin genes are single copy for alpha-skeletal and alpha-cardiac actin but multicopy for beta and gamma cytoskeletal genes: 3’ untranslated regions are isotype specific but are conserved in evolution. Mol. Cell. Biol. 3: 178.3. Smith, D. B., and Johnson, K. S. (1988). Single-step polypeptides expressed in E. coli as fusions with transferase. Gene 67: 31-40. Solomon, E., and Baker, D. F. (1989). genetic constitution of chromosome 319-337.

purification glutathione

of S-

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