Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene

We have isolated and sequenced part of a new gene of the tyrosine kinase family. This gene, called FLT3, has strong sequence similarities with members of a group of genes encoding growth factor receptors: FMS, KIT, and PDGFR. We have localized the human FLT3 gene to chromosome 13, band q12, and its mouse homolog to chromosome 5, region G.     

Molecular cloning, chromosomal mapping, and developmental expression of a novel protein tyrosine phosphatase-like gene

Protein tyrosine phosphatases (PTPs) mediate the dephosphorylation of phosphotyrosine. PTPs are known to be involved in many signal transduction pathways leading to cell growth, differentiation, and oncogenic transformation. We have cloned a new family of novel protein tyrosine phosphatase-like genes, the Ptpl (protein tyrosine phosphatase-like; proline instead of catalytic arginine) gene family. This gene family is composed of at least three members, and we describe here the developmental expression pattern and chromosomal location for one of these genes, Ptpla. In situ hybridization studies revealed that Ptpla expression was first detected at embryonic day 8.5 in muscle progenitors and later in differentiated muscle types: in the developing heart, throughout the liver and lungs, and in a number of neural crest derivatives including the dorsal root and trigeminal ganglia. Postnatally Ptpla was expressed in a number of adult tissues including cardiac and skeletal muscle, liver, testis, and kidney. The early expression pattern of this gene and its persistent expression in adult tissues suggest that it may have an important role in the development, differentiation, and maintenance of a number of different tissue types. The human homologue of Ptpla (PTPLA) was cloned and shown to map to 10p13-p14.     

Structure, expression and chromosomal location of the Oct-4 gene.

The map position of Oct-4 on mouse chromosome 17 is between Q and T regions in the Major Histocompatibility Complex (MHC), and it is physically located within 35 kb of a class I gene. Several Oct-4-related genes are present in the murine genome; one of them maps to chromosome 9. The genomic structure and sequence of Oct-4 determined in t-haplotypes reveals five exons, and shows no significant changes in the t12 mutant haplotype making it unlikely that Oct-4 and the t12 early embryonic lethal are the same gene. By in situ hybridization, detectable onset of zygotic Oct-4 expression does not occur until compaction begins at 8-cells, suggesting that there might be other regulatory factors responsible for initiating Oct-4 expression.     

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.     

Chromosomal localization of FLT4, a novel receptor-type tyrosine kinase gene.

A new human gene encoding a putative receptor-type tyrosine kinase (RTK) was isolated by screening a placenta cDNA library with a mouse Flt3 probe. The deduced amino acid sequence of the intracellular region of the molecule showed that it was strongly related to the FLT1 and KDR/FLK1 gene products and to a lesser degree to members of the class III RTKs: FMS/CSF1R, PDGFRA/B, KIT, and FLT3. The gene was named FLT4. Cosmid clones of the mouse Flt4 gene were isolated. The human gene was localized to bands q34-q35 of chromosome 5, i.e., slightly telomeric to the CSF1R/PDGRFB tandem of genes, and the mouse homolog to chromosome 11, region A5-B1.     

Coupled analysis of gene expression and chromosomal location

Microarray technology can be used to assess simultaneously global changes in expression of mRNA or genomic DNA copy number among thousands of genes in different biological states. In many cases, it is desirable to determine if altered patterns of gene expression correlate with chromosomal abnormalities or assess expression of genes that are contiguous in the genome. We describe a method, differential gene locus mapping (DIGMAP), which aligns the known chromosomal location of a gene to its expression value deduced by microarray analysis. The method partitions microarray data into subsets by chromosomal location for each gene interrogated by an array. Microarray data in an individual subset can then be clustered by physical location of genes at a subchromosomal level based upon ordered alignment in genome sequence. A graphical display is generated by representing each genomic locus with a colored cell that quantitatively reflects its differential expression value. The clustered patterns can be viewed and compared based on their expression signatures as defined by differential values between control and experimental samples. In this study, DIGMAP was tested using previously published studies of breast cancer analyzed by comparative genomic hybridization (CGH) and prostate cancer gene expression profiles assessed by cDNA microarray experiments. Analysis of the breast cancer CGH data demonstrated the ability of DIGMAP to deduce gene amplifications and deletions. Application of the DIGMAP method to the prostate data revealed several carcinoma-related loci, including one at 16q13 with marked differential expression encompassing 19 known genes including 9 encoding metallothionein proteins. We conclude that DIGMAP is a powerful computational tool enabling the coupled analysis of microarray data with genome location.     

Isolation and sequence analysis of a novel human tyrosine kinase gene.

Using v-abl probes, we have identified and cloned a novel fes/fps-homologous human cDNA, which we have designated FER (pronounced "fair"). This apparently full-length cDNA of 3.0 kilobases has an open reading frame of 2,466 base pairs and the capacity to encode a protein of 94,000 molecular weight. The cDNA contains regions homologous to the highly conserved tyrosine protein kinase domain of other oncogenes and growth factor receptors but lacks a clear transmembrane region, indicating that it encodes a tyrosine kinase of the nonreceptor type. The deduced amino acid sequence of FER resembles that of c-fes/fps. Our data indicate that the protein product of FER, p94FER, corresponds to a previously reported cellular phosphoprotein, NCP94, detected with a v-fps-specific antipeptide antiserum.     

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.     

Evolution of gene sequence in response to chromosomal location

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.     

Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location

The promoter region of the Neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (Münger, K., Germann, U. A., and Lerch, K. (1985) EMBO J. 4, 2665-2668). We therefore studied the regulation of expression of the N. crassa metallothionein gene in response to different metal ions (Cu2+, Cd2+, Zn2+, Co2+, and Ni2+) by Northern analysis. Only copper led to the induction of metallothionein mRNA. In N. crassa cultures inoculated and grown in copper-supplemented media, metallothionein mRNA appeared during the late logarithmic growth period (about 30 h after inoculation) and was detectable for a time period of more than 30 h. In response to copper shock, however, rapidly increasing amounts of metallothionein mRNA were detected within minutes after copper administration at any time in vegetatively growing mycelia of N. crassa. Maximum levels were detected about 1 h after addition of copper to the medium. The half-life time of the mRNA was estimated as 2.5 h. The amounts of copper metallothionein reach a maximum level at 3 h after induction and thereafter remain constant. The rapid induction by copper ions of metallothionein mRNA and metallothionein together with the remarkable stability of the native protein intracellularly suggest that this protein serves an important homeostatic role in the copper metabolism in this fungus. The structural gene of N. crassa metallothionein has been located on chromosome VI using restriction fragment-length polymorphisms as genetic markers.     

Genomic structure, tissue expression and chromosomal location of the LIM-only gene, SLIM1.

Human SLIM1 is a recently described gene of the LIM-only class encoding four and a half tandemly repeated LIM domains. LIM domains are double zinc finger structures which provide an interface for protein/protein interactions and are conserved in a variety of nuclear and cytoplasmic factors important in cell fate determination and cellular regulation. Here we report the structural organization, expression pattern and chromosomal localization of the human SLIM1 gene. SLIM1 was found to contain at least five exons with all four introns disrupting the coding region at a similar position relative to the respective complete LIM domains. Northern blot analysis confirmed strikingly high expression of SLIM1 in skeletal muscle and heart, with much lower expression observed in several other tissues including colon, small intestine and prostate. The SLIM1 gene was assigned to human chromosome Xq26 using fluorescence in situ hybridization.