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[Cancer Research 59, 4069-4074, August 1, 1999]
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[Cancer Research 59, 4069-4074, August 15, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

Identification of NKIAMRE, the Human Homologue to the Mitogen-activated Protein Kinase-/Cyclin-dependent Kinase-related Protein Kinase NKIATRE, and Its Loss in Leukemic Blasts with Chromosome Arm 5q Deletion1

Michael Midmer, Rizwan Haq, Jeremy A. Squire and Brent W. Zanke2

Departments of Medical Biophysics [M. M., J. A. S., B. W. Z.], Medicine [B. W. Z.], and Laboratory Medicine and Pathobiology [J. A. S.] and Institute of Medical Sciences [R. H., B. W. Z.], University of Toronto, and The Ontario Cancer Institute/Princess Margaret Hospital [M. M., R. H., J. A. S., B. W. Z.], Toronto, Ontario, Canada, M5G 2M9


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human acute leukemia and myelodysplasia are often associated with an interstitial deletion in chromosome arm 5q. The deleted region is hypothesized to contain tumor suppressor loci that are critical to the maintenance of normal hematopoiesis. We have identified NKIAMRE, a novel cyclin-dependent kinase-related molecule that is closely related to the rat serine/threonine kinase NKIATRE. Human NKIAMRE localizes to chromosome band 5q31.1, centromeric to the interleukin 9 locus and telomeric to IFN response factor-1. NKIAMRE was deleted at both alleles in 9 of 18 leukemic samples with chromosome band 5q31 abnormalities studied by fluorescence in situ chromosomal hybridization. NKIAMRE loss may be an important determinant of dysmyelopoiesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interstitial deletions involving the long arm of chromosome 5 [del(5q)] are commonly seen in human myeloid dysplastic disorders and leukemia (1) . Patients with such deletions as the sole karyotypic abnormality often have a relatively benign clinical course characterized by RAEB,3 modest leukopenia, macrocytic anemia, hypolobular megakaryocytes, and a low risk of transformation to acute leukemia (2 , 3) . Patients with disease that transforms to acute leukemia and those presenting with this disease de novo often display additional complex karyotypic abnormalities, suggesting that important genetic modulators of hematopoietic growth and differentiation may be lost as early events in all of these clinical conditions (4) .

To identify lost tumor suppressor genes in MDS and acute leukemia, investigators have attempted to define the minimally deleted region on chromosome arm 5q through the systematic comparison of a series of karyotypes derived from myeloid malignancies. Several groups have reported small conserved deletions of chromosome band 5q31.1 in a 2.4-Mb region flanked by the gene for IL-9 (centromeric) and the zinc finger transcription factor, EGR-1 (telomeric; Refs. 5 and 6 ). This region has been further refined, with the focus on a 1.0–1.5-Mb segment that is found between the locus markers D5S479 and D5S500 (7) . Other investigators have identified different deleted loci found either centromeric to the IL-9 gene, such as that containing the IRF-1 gene (8) , or telomeric to the EGR-1 gene, such as the site of the CSF1R (c-fms) gene (9) . Moreover, analyses of cytogenetic abnormalities in patients with non leukemic MDS have identified additional sites of deletion, such as the chromosome 5q13–5q21 region (10) or the more telomeric band 5q31.3 site (11) . This variable pattern of chromosome loss and the high density of hematopoietic growth regulating genes within chromosome 5q suggests that the loss of critical but variable combinations of genes may result in a clinical spectrum ranging from MDS to frank myeloid or lymphoid leukemia (1) .

In a separate report,4 we describe a rat MAP kinase-related signaling molecule that has sequence similarity to the CDK family of cell cycle regulators. This molecule, named NKIATRE after its putative cyclin-binding motif, is expressed exclusively in differentiated tissues and may have a role in growth and cell differentiation. Here, we report the identification of the human NKIATRE homologue, named NKIAMRE, which localizes to chromosome band 5q31.1. Because loss of this gene may have etiological relevance in acute leukemia, a series of primary leukemic blasts, selected for known deletion of chromosome band 5q31 on one chromosome homologue, were examined for loss of the second NKIAMRE allele. We report that both copies of NKIAMRE are deleted in a substantial proportion (34–70%) of cells in 9 of 18 samples with deletion at chromosome band 5q31. This data are consistent with a role for NKIAMRE, or a closely linked gene, in the etiology of human acute myelogenous leukemia.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning, PCR, and Sequencing.
cDNA library screening was performed as described previously (12) . In brief, 5 x 105 pfu of a {lambda}gt22 fetal heart cDNA library (gift from Dr. C. C. Liew, University of Toronto, Toronto, Ontario, Canada) was incubated with Y1090 bacteria and plated on 10 x 150 mm YT agar plates. Phage DNA from lysed bacteria cells was transferred to duplicate Hybond-N membranes (Amersham, Amersham, United Kingdom). After transfer, filters were denatured for 2 min in a solution of 1.5 M NaCl and 0.5 M NaOH and neutralized for 5 min in 1.5 M NaCl and 0.5 M Tris-HCl (pH 8). A 426-bp NotI-HindIII probe from a human EST (GenBank accession no. R21498), noted to be 89% identical to the previously isolated rat NKIATRE sequence (GenBank accession no. AF 112184), was radiolabeled with [{alpha}-32P]dCTP, using the Multiprime DNA labeling system (Amersham), and used as the probing sequence. Hybridization was performed overnight at 42°C followed by washing at 55°C in 0.1x SSC and 0.1% SDS. Filters were exposed to film overnight at -70°C with an intensifying screen. Purification of the hybridizing plaques was performed by secondary and tertiary screens, as described previously (12) .

Phage was purified by using Qiagen’s {lambda} phage DNA extraction kit. The insert DNA was amplified by PCR using vector-specific primers and cloned into the pCR2.1 vector (Invitrogen, San Diego, CA). The product was sequenced at the York University Core Molecular Biology Facility (Toronto, Ontario, Canada) using PCR-based methodology.

Isolation of Human Genomic NKIAMRE.
To facilitate subsequent chromosome hybridization studies, we hybridized rat NKIATRE cDNA to a commercial array of human BACs (generated by Dr. P. de Jong, Roswell Park Cancer Institute, Buffalo, NY).5 One identified clone, BAC 215P23, was partially sequenced using the NKIAMRE-specific primer ATGGAGATGTATGAAACC (sense, positions 1–18).

Patients.
The Princess Margaret Hospital annually treats over 100 patients with acute leukemia. Since 1990, patient material, including cryogenically preserved leukemic blasts, has been archived. Eighteen cases were randomly selected in which deletion of chromosome band 5q31 had been identified during routine cytogenetic analysis. Aliquots of methanol-/acetic acid-fixed cells derived from the original cytogenetic preparations were stored at 4°C prior to use for FISH studies. Good-quality interphase nuclei were present in all samples, whereas limited numbers of metaphase cells of adequate quality were present in some cases. For each patient, the clinical record was abstracted.

Chromosomal FISH.
FISH was performed in batches containing one normal donor bone marrow and three or four leukemic marrow samples using standard protocols (13) . Methanol-/acetic acid-fixed leukemic blasts were dropped onto coverslips, washed with a 3:1 mixture of methanol/acetic acid, and allowed to evaporate to encourage chromosome spreading. Slides were dehydrated in preparation for hybridization. Coincident hybridization was performed using the NKIAMRE locus-specific BAC 215P23 and with a hybridization control probe, BAC 42H215 (kindly provided by Dr. Barbara Beatty, Toronto), which had been mapped to human chromosome band 5p15 by the FISH Mapping Resource Center (The Ontario Cancer Institute). BAC 215P23 was labeled with biotin using the BioNick labeling system (Life Technologies, Inc.), whereas BAC 42H21 was labeled with DIG using the DIG-NICK Translation Mix (Boehringer Mannheim). Both probes were precipitated in the presence of salmon sperm DNA (Stratagene) and Cot-1 DNA (Life Technologies, Inc.). Slides and probes were denatured in the presence of formamide. Each probe (200 ng) was mounted onto slides followed by hybridization at 37°C overnight. The slides were washed with once in 70% formamide/2x SSC and twice in 2x SSC, followed by three washes in phosphate-buffered detergent. Between washes, double-color detection of the probes was performed. After washes 1 and 2, slides were mounted with anti-DIG (Boehringer Mannheim) and FITC-avidin (Oncor) antibodies, with DIG-antimouse (Boehringer Mannheim) and antiavidin (Oncor) antibodies after wash 3, and finally with rhodamine-anti-DIG (Boehringer Mannheim) and FITC-avidin (Oncor) antibodies after wash 4. Cells were stained with 4',6-diamidino-2-phenylindole to aid detection during fluorescence microscopy. Fifty cells were counted on each slide by two independent reviewers, blinded to sample identity.

Statistical Analysis.
Deletion of the NKIAMRE locus was evaluated by comparing the proportion of marrow cells having loss of either one or two BAC 215P23 signals in leukemic and in normal control samples. Each of these comparisons was done assuming two independent proportions with equal variance (14) . Statistical significance was determined by comparing the test statistic to the normal distribution, using a P < 0.05 cutoff.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Human NKIAMRE.
Several ESTs, which have sequence similarity to the rat NKIATRE gene, were found within the GenBank database.6 One unspliced 394-bp sequence (GenBank accession no. R21498) had 89% nucleotide identity over a 113-bp exonic region (Fig. 1Citation , underlined) and was used to screen a human fetal heart cDNA library. The longest library isolate contained the expected EST exonic sequence and had remarkable similarity to the rat NKIATRE ß isoform, with 79% amino acid identity overall. Comparison of this human library isolate to rat NKIATRE demonstrated that it lacked ~358 bp of 5' coding sequence. A further database search, however, identified three exons within a genomic PAC sequence having 88% nucleotide and 94% predicted amino acid identity to the 554 bp following the rat NKIATRE initiation codon (PAC H59; Lawrence Livermore National Laboratory, GenBank accession no. AC005354; Ref. 15 ). The distal 181 bp of the third coding exon was identical to the 5' terminus of our human cDNA library isolate, confirming that the cDNA was derived from a transcript of the genomic sequence. Using this genomic sequence, we were able to deduce the complete human coding sequence, which predicts a 455-amino acid serine threonine kinase that is 84% identical to the rat gene overall (Figs. 1Citation and 2Citation ).



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  		Fig. 1. The nucleotide and deduced amino acid sequences of NKIAMRE. Protein kinase consensus residues are shaded. The NKIAMRE motif and two sites of potential regulation, the SY duplex characteristic of the CDKs and the TDY MAP kinase-like regulatory region, are shown in boldface type. The sequence corresponding to EST R21498 is underlined.

 


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  		Fig. 2. Sequence alignment of NKIAMRE (human) compared to the NKIATRE ß isoform (rat) and the closest related kinases, KKIAMRE (human) and KKIALRE (human). Residues varying from NKIAMRE are highlighted. NKIATRE is 84% identical to NKIAMRE, whereas KKIAMRE and KKIALRE show 41 and 42% identity, respectively.

 
Like rat NKIATRE, the human sequence has features of both the MAP kinases and the CDKs. Like the MAP kinases, potential regulatory phosphorylation sites at threonine 158 and tyrosine 160 are observed in kinase subdomain VIII (16) . Threonine 158 coincides with a critical site of activating phosphorylation in the CDKs, whereas serine 14 and tyrosine 15 correspond to CDK sites of negative regulation (17) . Like rat NKIATRE, the human sequence has a putative cyclin-binding motif, although threonine has been replaced with methionine. In keeping with the naming convention of CDK-related molecules, this human gene has been named NKIAMRE. NKIAMRE has 84% amino acid identity to NKIATRE and 41 and 42% identity to the cdc 2-related kinases KKIAMRE and KKIALRE, respectively, which are the closest matches identified by Blast search6 (Fig. 2)Citation .

Isolation and Localization of Genomic NKIAMRE.
Prior to the recognition of NKIAMRE within the genomic PAC H59 sequence, we identified hybridization of rat NKIATRE to the human genomic BAC 215P23. Southern hybridization of the complete NKIATRE {alpha} sequence to EcoRI-digested gene fragments produced three hybridizing bands of 1.2, 7.6, and 3.7 kb. Direct partial sequencing of this clone demonstrated identity to NKIAMRE cDNA between 1 and 122 bp and to PAC H59 between 21,917 and 22,319 bp. FISH of labeled BAC 215P23 DNA to a normal human chromosome metaphase spread with coincident chromosomal G-banding confirmed the localization of NKIAMRE to chromosome band 5q31 (Fig. 3a)Citation . This localization is consistent with the reference location of PAC H59, as determined by FISH.7 PAC H59 has been oriented with respect to known genes, other genomic clones, and previously identified anonymous genomic markers on chromosome band 5q31 through the construction of an overlapping contig set spanning a 20-Mb region (strategy described on the Lawrence Berkley National Laboratory web site7 ). PAC H59 is ~1 Mb centromeric to the myeloid leukemia locus defined by Le Beau et al. (6) and 1.8 Mb telomeric to the IRF-1 locus, which is also implicated in acute leukemia (8) . This prompted us to evaluate NKIAMRE locus deletion in primary acute leukemic cells.



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  		Fig. 3. a, localization of NKIAMRE to 5q31 by FISH. Left, metaphase chromosome shows the NKIAMRE signal (green) localizing to band 5q31. Middle, 4',6-diamidino-2-phenylindole staining of the same chromosome. Right, schematic representation of chromosome 5 is shown. b, FISH performed on marrow of patient 8 with del(5q31) karyotype (see Table 1Citation ). All cells have two copies of the 5p15 telomeric control (red). Cells with two green signals are remaining normal marrow cells. Malignant cells have one green signal or absence of hybridization, if the remaining NKIAMRE allele has been lost.

 
NKIAMRE Locus Is Deleted in Acute Leukemia.
Deletions involving chromosome 5q are the most commonly observed genetic abnormalities in acute leukemia, occurring in 50% of cases presenting after prior chemotherapy and in 15% of cases arising de novo (18, 19, 20) . Because NKIAMRE localizes to this commonly deleted region, its monoallelic loss could simply reflect its proximity to other truly relevant loci. However, loss of both NKIAMRE loci, would have greater significance, suggesting that NKIAMRE, or a closely linked gene, is important for the generation or maintenance of the pathological state.

Eighteen methanol-/acetic acid-fixed samples of primary leukemic or dysplastic bone marrow were selected on the basis of an identified chromosomal aberration of band 5q31 detected by conventional G-banded analysis of metaphase spreads (Table 1)Citation . This collection contained 1 case of RAEB, 3 cases of acute lymphocytic leukemia of the L2 subtype, and 14 cases of acute myelogenous leukemia. Of the 14 acute myelogenous leukemia cases, 8 had a prior clinical history or marrow morphological evidence of MDS.


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  		Table 1 Clinical and karyotypic features of study patientsa

 
To evaluate potential biallelic loss of NKIATRE within these samples, we hybridized slides derived from cryopreserved interphase nuclei to biotin-conjugated BAC 215P23 and detected with FITC-conjugated avidin (21) . In all experiments, hybridization efficiencies were monitored within and between individual samples by simultaneously scoring hybridization signals from the BAC 42H21, a single-copy genomic probe mapping to chromosome band 5p15. BAC 42H21 was labeled with DIG and detected by rhodamine-conjugated anti-DIG antibody, producing red fluorescence, distinct from the green fluorescence of BAC 215P23 hybridization (Fig. 3b)Citation . Analysis was restricted to cells having two chromosome band 5p15 (probe 42H21) hybridizing signals.

Hybridization efficiencies of >90% were observed for both probes when FISH was performed on nuclei from diploid control bone marrow samples, indicating that each probe had comparable detection sensitivities (data not shown). Similarly, the chromosome band 5p15 control probe (42H21) exhibited two signals in >90% of nuclei when hybridized to leukemic nuclei. Table 2Citation shows the percentage of nuclei in which homozygous, hemizygous, and no deletion of BAC 215P23 (NKIAMRE) was apparent. As expected, most leukemic cells had at least one deleted NKIAMRE locus (13 of 18). In five samples, the proportion of cells having loss at this locus did not differ from controls, suggesting either inaccuracies in the karyotypic analysis of these cases or significant admixture of normal marrow hematopoietic or stromal cells, which prevented the detection of statistical significant deletion. Of 13 samples having BAC 215P23 loss, 9 displayed statistically significant biallelic NKIATRE deletion. Within these individual samples, the percentage of cells having biallelic deletion was quite variable, ranging from 34 to 70%.


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  		Table 2 NKIAMRE fluorescence by in situ chromosomal hybridization in leukemic samples and normal controlsa

 
The clinical records of patients were reviewed to determine whether NKIAMRE deletion was associated with a distinct clinical profile. Within this small sample size, patients with biallelic NKIAMRE deletion were not distinguished by age at diagnosis, sex, French-American-British leukemic subtype, previous chemotherapy, antecedent MDS, or survival after diagnosis (Table 1)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The study of recurrent chromosomal loss in human malignancies has identified new tumor suppressor genes and highlighted the importance of established negative growth regulators (22, 23, 24, 25) . Interstitial deletion in human chromosome arm 5q is a recurrent abnormality associated with acute leukemia and with myelodysplastic hematopoiesis that is characterized by megaloblastic anemia and dysmegakaryopoiesis (2 , 4 , 18) . Such patients who progress to acute leukemia and who present with chromosome 5q arm abnormalities have a particularly poor prognosis (26) . These observations have directed attention to the long arm of chromosome 5 as a likely tumor suppressor locus that may be lost early in the development of malignant hematopoiesis because accumulation of additional karyotypic abnormalities characterizes diseases of higher grade (2) . In patients with chromosome arm 5q deletion, progression from dysmyelopoiesis to acute leukemia could also result through mutation or structural rearrangement of the single remaining tumor suppressor locus, analogous to the molecular pathogenesis of retinoblastoma (2) .

Despite much interest in the genomic characterization of chromosome 5q, a critical tumor suppressor gene has not yet been identified. Through the molecular analysis of selected patients with small 5q deletions, several groups have attempted to define the essential putative tumor suppressor locus. Through the use of high-resolution banding techniques, chromosome band 5q31 has been defined as the most uniformly deleted region in 91 of 93 leukemic patients with chromosome arm 5q loss (26) . A study focusing on polymorphisms of the anonymous genomic marker, D5S89, identified loss of heterozygosity within this region in five patients with partial chromosome arm 5q deletion (27) . The D5S89 sequence, encompassed within a 300-kb yeast artificial chromosome clone, maps telomeric to the IL-9 gene but centromeric to the EGR-1 gene, thus excluding involvement of the loci for IL-3, IL-4, IL-5, and granulocyte macrophage colony-stimulating factor, which are found centromeric to this site. Using contiguous PAC clones spanning the region from IL-9 to the marker D5S166, we identified, through FISH analysis of 28 patients with cytogenetically detectable loss of band 5q31, an overlapping 1–1.5-Mb region of consistent deletion, found between markers D5S479 (centromeric) and D5S500 (telomeric), encompassing the EGR-1 gene (Ref. 7 ; Fig. 4Citation ). In contrast, a study of two patients with MDS and uncommonly small 5q deletions used pulse-field gel electrophoresis to map chromosome arm 5q deletion breakpoints to a nonoverlapping site telomeric to the EGF-1 locus (28) . In addition, both copies of the IRF-1 locus, which are found centromeric to all other sites of minimal deletion, were deleted in 5 of 19 patients with chromosome arm 5q-associated myeloid dyscrasias in one study (8) . These observations suggest that phenotypically uniform myeloproliferative disorders may have a complex and variable polygenic etiology.



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  		Fig. 4. Position of NKIAMRE on human chromosome band 5q31.1 relative to known genes and genomic markers. The position of human PAC H59 relative to known genes has been determined through the construction of an overlapping contig set by the Lawrence Berkley National Laboratory Genomics Group.7 NKIAMRE, which is partially found within PAC H59, is telomeric to the IRF-1 gene and centromeric to the IL-9 gene.

 
Here, we describe the identification and chromosomal location of NKIAMRE, a human homologue of the rat NKIATRE gene, which maps to chromosome band 5q31. A detailed assignment of NKIAMRE within this chromosomal band was possible through the recognition that its coding region was partially contained within the 28-kb PAC H59, which derives from a 20-Mb YAC contig localizing to chromosome band 5q31.7 NKIAMRE lies immediately telomeric to the TCF1 locus and is found ~2 Mb centromeric to the IL-9 gene (Fig. 4)Citation . Although NKIAMRE lies centromeric to the minimally deleted regions described by Zhao et al. (29) , Boultwood et al. (28) , and Nagarajan et al. (27) , it lies ~2.5 Mb telomeric to the IRF-1 locus, a site implicated in myeloid dyscrasias as described above. Given the likely polygenic etiology of leukemia associated with chromosome arm 5q deletion, we studied NKIAMRE loss in a sample of patients with acute myeloid and lymphoid leukemia. Using FISH, leukemic cells with a single copy deletion of the chromosome band 5q31 segment were evaluated for microdeletion of the remaining homologous locus. We observe statistically significant NKIAMRE deletion in half of leukemic cell populations. Of interest, marked heterogeneity within individual leukemic samples was noted with the proportion of cells having two-copy loss varying from 34 to 70%. A similar observation has been made with respect to loss of the IRF-1 domain in karyotypically similar group of patients (8) . In this study, leukemic cells missing both IRF-1 hybridization domains were seen in 6 of 11 samples tested, with individual proportions as high as 20%. These subpopulations of cells may be emerging leukemic clones, selected for absence of a dose-sensitive tumor suppressor locus. Although the evolution of leukemic subpopulations with increased karyotypic complexity signals a poor clinical prognosis, clinical outcome was not statistically worse for patients lacking the NKIAMRE locus in a significant proportion of their malignant cells (30 , 31) .

NKIAMRE, a 455-amino acid protein with predicted molecular weight of 52,000, like rat NKIATRE, has strong sequence similarity to both the CDKs and to the MAP kinases. Similar to these two groups of kinases, the translation product of NKIATRE contains the sequences DIKPEN in kinase subdomain VI (residues 125–130) and ATRWYR in kinase subdomain VIII (residues 162–167), which are strong predictors of serine/threonine kinase activity (32) . NKIATRE expression is restricted to differentiated primary tissues and expression in rapidly dividing hematopoietic cell cultures is undetectable. NKIATRE, which localizes to the cell cytoplasm, may, therefore, have a role in cell growth or differentiation control through the modulation of signaling cascades. Loss of one copy of NKIAMRE may cause myelocytic differentiation abnormalities, whereas homozygous loss may be associated with higher-grade disease.

We have identified a new human gene mapping to chromosome band 5q31 and have demonstrated deletion of both loci in a sample of leukemic patients with cytogenetically detectable hemizygous chromosome arm 5q deletion. Although NKIAMRE loss may contribute to the transformed phenotype of our study population, loss of closely linked genes may also contribute or be solely responsible for the transformed phenotype. The role of NKIAMRE in a broader leukemic population awaits definition through more extensive FISH studies of the region, together with mutational and protein expression analysis of NKIAMRE.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Medical Research Council of Canada-Pharmaceutical Manufacturers Association of Canada program in collaboration with BioChem Pharma (Laval, Quebec, Canada). Back

2 To whom requests for reprints should be addressed, at The Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada, M5G 2M9. Phone: (416) 946-2961; Fax: (416) 946-6546. E-mail zanke{at}oci.utoronto.ca Back

3 The abbreviations used are: RAEB, refractory anemia with excess blasts; MDS, myelodysplasia; EGR, early growth response; IL, interleukin; IRF-1, IFN response factor-1; MAP, mitogen-activated protein; CDK, cyclin-dependent kinase; EST, expressed sequence tag; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; DIG, digoxigenin; PAC, P1 artificial chromosome. Back

4 R. Haq, S. Randall, M. Midmer, K. Yee, B. Iafrate, and B. Zanke. NKIATRE, a novel alternatively spliced cdc-2-related kinase, submitted for publication. Back

5 http://bacpac.med.buffalo.edu. Back

6 http://www.ncbi.nlm.nih.gov/. Back

7 http://www-hgc.lbl.gov/biology/mapping.html#Chrom5. Back

Received 2/22/99. Accepted 6/10/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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