Genes In Human Tissues Research Articles

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Quantitative expression of the HERV-W env gene in human tissues

Human endogenous retroviruses HERV-W families have been identified from monozygotic twin pairs with schizophrenia and patients with multiple sclerosis. Identification of retroviral RNA in the cerebrospinal fluids and brains of individuals with schizophrenia indicated that the transcriptional activation of the HERV-W elements within the central nervous system could be associated with the brain diseases. Here, we examined the expression of the HERV-W env gene in tumor/normal adjacent tissues and various areas of brain tissues. In silico expression data indicated that 14 complete HERV-W families from human chromosomes 1, 2, 4, 7, 8, 11, 13, 15, and Y are randomly expressed in various cancer tissues. HERV-W env transcripts did not show significant differences among the human tumor/normal adjacent tissues (colon, liver, uterus, breast, and stomach). Quantitative real-time RT-PCR analysis indicated that strong expression of the HERV-W env gene was detected in the cerebral cortex and pons of the human brain.

M02-04: Genetic markers of tumor response

Recently, individualized and tailored molecular targeted therapy was investigated for overcoming the limit of the present modalities of cancer treatment. Detoxification enzymes (NQO1, GSTP1, MPO and MnSOD), DNA repair gene, XRCC1/2, ERCC1 and the others are studied, and the expression or mutations of these genes in human tissues are reported world-widely as a factor related to risk of cancer in lung or other sites. Furthermore, some reports investigated the difference of treatment results by expression or mutations of these genes with chemotherapy or radiation therapy. Firstly, most studies have focused on the DNA repair gene, because DNA repair enzyme is critical for the protection of the genome from carcinogenic exposure and the prevention of some types of cancer. It is possible that the DNA repair mechanism plays a key role in the response of cancer cells to ionizing radiation, because the cytotoxic effect of radiation is mediated by the single or double strand breaks in the DNA. There are 4 major DNA repair mechanisms for maintaining the genomic integrity against damage caused by mutagens: mismatch repair, base excision repair, nucleotide excision repair, and double strand break repair. The X-ray repair cross-complementing Group 1 (XRCC1) plays an important role in the DNA base excision repair pathway, giving the possibility that this enzyme has some relationship with the response to radiotherapy. Secondly, a few studies have revealed that NAD(P)H:quinine oxidoreductase (NQO1, previously called as DT-diaphorase) is manifested within epithelium and endothelium in human tissues, and is also at many tumors, particularly NSCLC, originated from epithelial cell. NQO1 is two-electron reductases which catalyze more toxic quinines to less toxic hydroquinones, so it was revealed that NQO1 may play a major role in cytoprotection and chemoprevention. NQO1 also prevents the generation of reactive oxygen (hydrogen peroxide, superoxide) to human tissue, and is revealed as scavenger of supreoxide. Radiation therapy gives cytotoxic damage to human tissue via pathway of direct or indirect DNA breakage and indirect pathway breaks DNA strands by secondary effect of free radical produced by radiation particle that a potent inducer of NQO1 expression in human tissues. β-Lapachone (3,4-dihydro-2,2-dimethyl1-2H-naphthol[1,2-b] pyran-5,6-dione) (β-Lap) was originally isolated from the bark of Lapacho tree (Tabebuia avellanedae) growing in South America. This drug has attracted considerable interest in recent years because of its potent cytotoxicity against various cancer cell lines through a mechanism that works independent of the cell cycle or p53 status. β-Lap induced cell death in vitro has been previously attributed to activation or inhibition of Topoisomerase II-α, inhibition of Topoisomerase II-α, and suppression of NF-ϰB activity. However, more recent studies have clearly indicated that none of these changes were the key determinant of cell death caused by β-Lap, particularly in vivo. Recently, NQO1 has been reported to be a key player in β-Lap induced cell death. NQO1 catalyzes a two-electron reduction of β-Lap to the hydroquinone form of β-lap, i.e., β-Lap(HQ), using NADH or NAD(P)H as electron donors. The resulting β-Lap(HQ) is unstable and consequently reoxidizes in the presence of O2 to original oxidized β-Lap, causing a futile cycling between the quinone and hydroquinone forms. This futile cycling of the drug causes a progressive depletion of NADH or NAD(P)H levels. We observed that IR sensitizes cancer cells to β-Lap by causing a long-lasting elevation of NQO1 activity. It thus appeared that the synergistic interaction of ionizing radiation (IR) and β-Lap in killing cancer cells was due to an increase in cellular susceptibility to β-Lap, probably in addition to β-lap induced radiosensitization. Importantly, the NQO1 level in many human tumors is markedly greater than this in normal tissues. The fact that NQO1 level in tumors can be increased further by IR suggests that the combination of β-Lap and radiotherapy may be a potentially effective approach to selectively damage tumors relative to normal tissue. The combination of β-Lap and radiotherapy is a potentially effective regimen for the treatment of human cancer. It is possible that genetic variations in genes mentioned above can affect the treatment result after radiation therapy, because its expression or mechanism is related to ionizing radiation. Single nucleotide polymorphisms (SNPs) make up about 90% of human genetic variations. We tried to investigated that the difference in enzymatic activity by genetic polymorphisms could affect the treatment response and might be used as genetic marker of tumor response after radiation therapy.

Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells

Long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) have been reported to serve as alternative promoters in functional genes. The GSDML (gasdermin-like protein) gene located on human chromosome 17q21 has been found to be an oncogenomic recombination hotspot. Here, we identified the LTR element of HERV-H with reverse orientation as an alternative promoter of the GSDML gene and analyzed its expression pattern in human tissues and cancer cells. A reporter gene assay of the promoter activity of the LTR on the GSDML gene in human cancer cell lines (HCT-116 and HeLa) and a kidney cell line (Cos7) of African green monkey indicated that the LTR promoter with reverse orientation had stronger promoter activity than forward one. The transcripts of this LTR-derived promoter were widely distributed in various human tissues and cancer cells, whereas the transcripts of the cellular promoter were found only in stomach tissues and some cancer cells (HCT116, MCF7, U937, C-33A, and PC3). These findings suggest that the LTR element on the GSDML gene was integrated into the hominoid lineage and acquired the role of transcriptional regulation of human tissues and cancer cells.

Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue

BackgroundLow density arrays (LDAs) have recently been introduced as a novel approach to gene expression profiling. Based on real time quantitative RT-PCR (QRT-PCR), these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to QRT-PCR. We have now evaluated LDAs as a means of determining the expression of multiple genes simultaneously in human tissues and cells.ResultsComparisons between LDAs reveal low variability, with correlation coefficients close to 1. By performing 2-fold and 10-fold serial dilutions of cDNA samples in the LDAs we determined a clear linear relationship between the gene expression data points over 5 orders of magnitude. We also showed that it is possible to use LDAs to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of 1 × 102 – 1 × 106 copies. Furthermore, the data generated by the LDA from a cell based pharmacological study were comparable to data generated by conventional QRT-PCR.ConclusionLDAs represent a valuable new approach for sensitive and quantitative gene expression profiling.

Endogenous retrovirus-related sequences provide an alternative transcript of MCJ genes in human tissues and cancer cells

The MCJ gene is a member of the DNAJ family, and its transcriptional event is controlled by methylation of the CpG island. In our study, we found LTR33 and LTR7 elements provided an alternative transcript within the MCJ gene. To detect different expression patterns between the originally reported MCJ transcript and the LTR-related transcript, we performed a RT-PCR approach using various human tissues and cancer cells. The original MCJ transcript was detected in human tissues and cancer cells, whereas the LTR-related transcript was only revealed in some cancer cells (HCT106, MCF-3, TE-1, Hela, and CCHM). We also performed a PCR analysis to compare the insertion lineage of the LTR elements with the genomic DNAs of primates, indicating that those LTR33 and LTR7 elements of HERV-H have been integrated into the primate genome at different times. Taken together, we suggest that HERV-related elements trigger transcriptome diversification during primate evolution.

GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues

Quantitative gene expression data are often normalized to the expression levels of control or so-called "housekeeping" genes. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the cells or tissues under investigation. Although exceptions to this assumption are well documented, housekeeping genes are of value in fully characterized systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. To investigate the value of GAPDH as a housekeeping gene in human tissues, the expression of GAPDH mRNA was measured in a panel of 72 different pathologically normal human tissue types. Measurements were obtained from 371,088 multiplexed, quantitative real-time RT-PCRs with specific target genes. Significant differences in the expression levels of GAPDH mRNA were observed between tissue types and between donors of the same tissue. A 15-fold difference in GAPDH mRNA copy numbers was observed between the highest and lowest expressing tissue types, skeletal muscle and breast, respectively. No specific effect of either age or gender was observed on GAPDH mRNA expression. These data provide an extensive analysis of GAPDH mRNA expression in human tissues and confirm previous reports of the marked variability of GAPDH expression between tissue types. These data establish comparative levels of expression and can be used to add value to gene expression data in which GAPDH is used as the internal control.

Methylation and expression of the lactoferrin gene in human tissues and cancer cells.

The lactoferrin gene promoter contains GC-rich regions that harbor consensus sequences for a variety of transcription factors. Previous work in our laboratory has demonstrated a link between methylation at the CpG sites of the mouse lactoferrin gene promoter and the level of its expression. The current work investigates the methylation profile in three regions of the human lactoferrin gene by bisulfite genomic sequencing. In addition, the methylation profiles of normal leukocyte DNA, and leukemia cell line and patient DNA were compared. The three regions are located at the -504/-190, which includes the estrogen response element, the -282/+271 which contains the lactoferrin promoter and the +1087/+1476, a region within the first intron that has the alternative delta lactoferrin promoter. Differential methylations were found within all the regions. Increased methylation at the CpG sites and the presence of non-CpG methylation of the lactoferrin promoters were found in the cancer samples.

Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR2

ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

Characteristic expression of aryl hydrocarbon receptor repressor gene in human tissues: Organ-specific distribution and variable induction patterns in mononuclear cells

To investigate the expression of aryl hydrocarbon receptor repressor (AhRR) and related molecules in various tissues and the effects of aromatic hydrocarbons (AHs) on their expression, we developed a reliable technique of quantification of human AhRR as well as aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) and cytochrome P450 1A1 (CYP1A1) mRNA by real-time TaqMan PCR method. First, we examined the expression of these genes in human adult or fetal tissues. The levels of AhRR expression were extremely high in testis, very high in lung, ovary, spleen and pancreas from adults, whereas those were low in those from fetuses. On the other hand, CYP1A1 expression was extremely high in lung, and AhR and ARNT were ubiquitously expressed in almost all tissues. Second, we compared the expression levels of these genes in mononuclear cells (MNCs) from various sources. Comparison of the basal expression levels of these genes in MNCs demonstrated that MNCs from umbilical cord blood showed higher AhRR or CYP1A1 expression than those from adults. The induction of AhRR or CYP1A1 expression by 3-methylcholanthrene (3-MC) was observed in MNCs from adults but not from umbilical cord blood. Consequently, there existed characteristic differences in the basal levels of AhRR and CYP1A1 expression in MNCs, as well as in their inducibility by 3-MC among MNCs from various types of human bloods. These results will provide basic information for a possible application of AhRR and CYP1A1 measurements to evaluate AH exposure in vivo.

Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP

The stathmin family consists of phosphoproteins highly conserved in vertebrates and thought to be implicated in the development and functional regulation of various organs, most notably the nervous system. This family includes stathmin, SCG10, SCLIP, and RB3, phosphoproteins that are related by structural and functional homologies. They all sequester tubulin and interfere with microtubule dynamics, a property due to their shared stathmin-like domain. Little is known about the expression of the stathmin gene family in humans. Herein, we describe for the first time, for a collection of human tissues, the expression of each member of this family, using real-time quantitative RT-PCR. We found that stathmin is ubiquitously expressed, whereas SCG10 and RB3 are neural enriched, expression patterns similar to those reported for other mammals. Surprisingly, SCLIP, whose expression is thought to be neural-specific, exhibits a broader tissue distribution. Analyses of the SCLIP gene (approved symbol STMN3) show that it contains several NRSE-like elements that display low or no affinity for the cognate binding protein NRSF. The substantial expression of SCLIP in most tissues points out a novel function for this protein outside the nervous system and raises the possibility that its coexpression with stathmin could provide some degree of functional redundancy.

Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells

Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5′ flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the −217/−10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A–D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.

Expression Profiling of Human Sulfotransferase and Sulfatase Gene Superfamilies in Epithelial Tissues and Cultured Cells

The bioavailability of drugs administered topically or orally depends on their metabolism by epithelial enzymes such as the cytosolic sulfotransferases (SULT). Reverse transcriptase-polymerase chain reaction (RT-PCR) methods were established to detect expression of 8 SULT genes and 4 arylsulfatase (ARS) genes in human tissues of epithelial origin and in cultures of normal and transformed (cancer) cells. The results indicate: (i) SULT 1A1, 1A3, ARSC, and ARSD genes are ubiquitously expressed; (ii) expression is frequently similar between cell lines and corresponding tissues; (iii) SULT gene expression in normal cultured cells is generally comparable to the expression in associated transformed (cancer) cell lines; (iv) SULT 1A1 promoter usage is mainly tissue specific; however, both promoters are frequently used in SULT 1A3 expression; and (v) the expression profile of SULT 1A1, 1A3, 1E1, and 2B1a/b suggests that one or more of these isoforms may be involved in the cutaneous sulfoconjugation of minoxidil and cholesterol.

Expression of the neurofibromatosis type 2 gene in human tissues.

The neurofibromatosis Type 2 tumor suppressor gene is implicated in the hereditary tumor syndrome NF2, hallmarked by bilateral vestibular schwannomas, meningiomas, and ocular non-neoplastic features. The gene product has characteristics of a membrane cytoskeleton-linking protein but the mechanism of tumor suppression by the NF2 protein remains to be elucidated. The NF2 gene is widely expressed in mouse and rat tissues. In humans, most of the expression data have accumulated through Northern blot analysis, RT-PCR and, more recently, Western blot analysis, providing information on whole tissues and organs rather than on specific cell types. We report here an extensive survey of NF2 gene expression in human tissues using a combination of mRNA in situ hybridization (mRNA ISH) and immunohistochemistry (IH) with a panel of monoclonal antibodies (MAbs) supplemented by tissue immunoprecipitation experiments with affinity-purified polyclonal antibodies. Expression was observed in many different cell types, most of which appear functionally normal in individuals affected by NF2. Surprisingly, expression could not be consistently documented in Schwann cells and arachnoidal cells by IH or by mRNA ISH in formalin-fixed tissue. However, consistent immunostaining of Schwann cells was seen in frozen sections. (J Histochem Cytochem 47:1471-1479, 1999)

Differentially expressed messenger RNA isoforms of the human estrogen receptor-alpha gene are generated by alternative splicing and promoter usage.

The isolation and characterization of several new human estrogen receptor-alpha (hERalpha) mRNAs are described. Together with those previously identified, they give rise to a total of six hERalpha mRNA isoforms (A-F hERalpha mRNAs). Produced from a single hERalpha gene by multiple promoter usage, all these transcripts encode a common protein but differ in their 5'-untranslated region as a consequence of alternative splicing of five upstream exons (1B-1F). RT-PCR and S1 nuclease mapping analysis of these different hERalpha mRNA isoforms revealed a differential pattern of expression of the hERalpha gene in human tissues and cell types. The A hERalpha mRNA is the main isoform detected in mammary glands or in the tumor cell lines derived from this tissue. In endometrium, the predominant forms are the A and C hERalpha mRNA isoforms, whereas the C and F hERalpha mRNA isoforms are the major forms detected in ovary. Finally, high levels of the E hERalpha mRNA isoform are restricted to the liver with an increased expression in females. Taken together, our results demonstrate that the hERalpha gene is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner.

Cloning, Expression Pattern, and Chromosomal Assignment to 16q23 of the Human γ-Adaptin Gene (ADTG)

Adaptins are important components of clathrin-coated vesicles transporting ligand–receptor complexes from the plasma membrane or from thetrans-Golgi network to lysosomes. Adaptins, together with medium and small subunits, form a heterotetrameric complex called an adaptor, whose role is to promote the formation of clathrin-coated pits and vesicles. We present the cloning and sequencing of the human γ-adaptin cDNA (HGMW-approved symbolADTG) consisting of 3723 bp with an open reading frame capable of encoding a protein of 825 amino acids, 98.9% identical to the mouse protein. Northern blot analysis of the mouse and human γ-adaptin genes revealed a ubiquitous and abundant expression, except in human adult lung. Using a monochromosomal somatic cell hybrid panel and fluorescencein situhybridization, we mapped this gene to human chromosome 16q23, which is syntenic with mouse chromosome 8, band D. In addition, we localized genes for two other components of the AP-1 adaptor, i.e., the medium (AP47) and small (AP19) subunits, to chromosomes 19 and 7, respectively. Expression analysis of these genes in human tissues revealed ubiquitously expressed transcripts of approximately 2.5 and 1.5 kb, respectively.

Partial cloning and differential expression of ryanodine receptor/calcium-release channel genes in human tissues including the hippocampus and cerebellum

Cellular Ca 2+ signalling is an important factor in the control of neuronal metabolism and electrical activity. Although the roles of Ca 2+-release channels are well established for skeletal and cardiac muscle, less is known about their expression and roles in the central nervous system, especially in the human brain. We have isolated partial complementary DNAs derived from the human ryanodine receptor Ca 2+-release channel genes ( ryr1, ryr2 and ryr3), and examined their expression in the human hippocampus and cerebellum. For comparison, we have included in our analysis an inositol trisphosphate Ca 2+-release channel type I complementary RNA probe. All four messenger RNAs show widespread distribution in the human hippocampus, where ryr2 is the most abundant isoform, and all four are expressed in the human cerebellum. However, striking differences were seen between ryr and inositol trisphosphate Ca 2+-release channel type I complementary RNA expression in the cerebellum, with inositol trisphosphate Ca 2+-release channel type I messenger RNA being largely restricted to, and very highly expressed, in Purkinje cells, whereas ryr1, ryr2 and ryr3 were all expressed predominantly in the granular layer. The widespread expression of ryr isoforms in the human hippocampus and cerebellum suggests that ryanodine receptor proteins may have a central role in Ca 2+ signalling and Ca 2+ homeostasis in the human central nervous system. These may include roles in fundamental processes like synaptic plasticity. Furthermore, these Ca 2+-release channels may be involved in pathogenic processes such as excitotoxicity, where excessive rises in intracellular Ca 2+ concentration mediate neuronal cell death.

Alternative splicing in the coding region of human aromatic l-amino acid decarboxylase mRNA

Total RNA from human neuroblastoma cells (SK-N-SH) was reverse transcribed and amplified using primers specific for aromatic l-amino acid decarboxylase (AADC). Two polymerase chain reaction (PCR) products were observed following agarose electrophoresis. Cycle sequencing of the PCR products revealed the larger fragment (414 bp) to be identical to the published human cDNA sequence (Type I). Sequencing of the smaller band (300 bp) demonstrated a form missing exon three (Type II). Both types of the mRNA were colocalized in human brain regions (gray matter and white matter) and other human tissues (liver, kidney, adipose, heart, adrenal gland and keratinocytes). The relative concentrations varied in each tissue studied but specific neuronal or non-neuronal patterns were not apparent. The study demonstrates alternative splicing within the coding region of the human AADC mRNA and the results suggest the possibility that two proteins are derived from the AADC gene in human tissues.

Expression of the Endothelin-Converting Enzyme Gene in Human Tissues

Based on the cDNA sequence of the human and bovine endothelin converting enzyme (ECE-1) we have developed a novel reverse transcription polymerase chain reaction to investigate the tissue expression of this gene. We were able to specifically detect the gene mRNA starting from very limited amount of tissue in all human as well as bovine tissues examined. Thus, our results confirm a widespread expression of the ECE-1 gene in human tissues, in keeping with the findings in other species, and suggest a major biological role of ECE-1.

Characterization and chromosomal localization of the human homologue of a rat AMP-activated protein kinase-encoding gene: a major regulator of lipid metabolism in mammals

AMP-activated protein kinase (AMPK) phosphorylates and inactivates acetyl-CoA carboxylase and β-hydroxy β-methylglutaryl-coenzyme A (HMG-CoA) reductase which are the major enzymes involved in fatty acid and lipid biosyntheses. The AMPK gene from rat ( rAMPK) has recently been cloned [Carling et al. J. Biol. Chem. 269 (1994) 11442–11448]. In order to study the structure and function of the human AMPK gene (h AMPK), we have cloned the gene, and report in this communication its nucleotide (nt) sequence, tissue distribution and chromosomal location. Our results show that the ORF of h AMPK encodes 552 amino acids (aa) (62.250 kDa) and is highly conserved with r ampk with identities of 97.3 and 90% at the aa and nt levels, respectively. The h AMPK gene bears homology to a yeast protein kinase-encoding gene ( snf1) that regulates carbohydrate metabolism, and also with three other genes encoding SNFl-like kinases from different plant species, namely Arabidopsis thaliana, Hordeum vulgare and Secale cereale. As determined by fluorescent in situ hybridization of a human metaphase chromosome spread, h AMPK maps to chromosome lp31. The size of the h AMPK transcript is 8.5 kb and the transcription start point ( tsp) is located approx. 46 bp upstream from the ATG codon. While 10–15% of AMPK is alternatively spliced in most tissues of the rat, our RT-PCR analyses of the h AMPK mRNA did not reveal the presence of any alternatively spliced form of the gene in human tissues. An interesting aspect of AMPK is that its expression, unlike in rat liver, could not be detected in human liver, and thus the purported role of the gene in controlling fatty-acid synthesis in the human liver remains to be determined.