The gene for human chromogranin A (CgA) is located on chromosome 14

Chromogranin A (CgA) is a protein that is present in most neuroendocrine tissues and is co-secreted with their resident hormones. We have assigned the CgA gene to human chromosome 14 by hybridization of a CgA cDNA probe cloned from a cDNA library of human medullary thyroid carcinoma cells to spots of individual human chromosomes flow-sorted onto nitrocellulose filters. Southern analysis of human genomic DNA with the same probe revealed only 1-3 restriction bands. These studies indicate that the CgA gene is probably single copy and not a member of a dispersed, multigene family. The CgA gene is not co-localized with the genes of any of the CgA-associated hormones.     

Cloning and characterization of two alternatively spliced rat alpha-amidating enzyme cDNAs from rat medullary thyroid carcinoma

The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.     

Evidence for transcription and potential translation of the human 1.9 kb HindIII repetitive element.

Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes.     

Selective processing of chromogranin A in the different islet cells in human pancreas.

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the four major neuroendocrine cell types of the human pancreas by using double immunofluorescence techniques. The antibodies raised to the N-terminal and midportions of CgA showed, on the whole, stronger immunoreactivity than did the C-terminal antibodies, with a few exceptions. Often the immunoreactivity was stronger in glucagon cells. Insulin cells expressed immunoreactivity to all region-specific antibodies, but glucagon cells were nonreactive to two antibodies. Somatostatin cells reacted only with the C-terminal antibodies (amino acid sequences CgA 411-424), while PP cells were stained with four CgA region-specific antibodies between amino acid sequences 63-195. The cause of these differences may be that the CgA molecule is cleaved, partly masked, or partly translated from CgA mRNA. Microwave treatment improved only the staining with the CgA 361-372 antibodies, which indicates that masking is not the sole or entire cause. Our findings may indicate that the CgA molecule is cleaved in different ways in the various pancreatic endocrine cell types, giving rise to a variety of biologically functional fragments.     

A simple, effective method for the construction of subtracted cDNA libraries

A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.     

Molecular cloning of porcine alveolar macrophage-derived neutrophil chemotactic factors I and II; identification of porcine IL-8 and another intercrine-alpha protein.

Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung. Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo. We report here the cloning of the full-length cDNAs which code for each protein. Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)(+)-selected mRNA. Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins. These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs. Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants. AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8. By contrast, AMCF-II has no obvious human homologue. AMCF-II shares 53% identity with human neutrophil activating peptide 2. Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67%. AMCF-II may represent a new member of the intercrine-alpha family of neutrophil chemoattractants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of SMART-generated cDNA for gene expression studies in multiple human tumors

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.     

Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Identification of receptor genes in renal cell carcinoma associated with angiogenesis by differential hybridization technique

To screen the receptor genes in renal cell carcinoma (RCC) associated with angiogenesis, we performed differential hybridization of the cDNA library of membrane-type protein tyrosine kinases (mPTKs). Three thousand plaques of a mPTKs-enriched cDNA library were screened with mPTKs mixture probes produced from hypervascular RCC tissues and RCC cell lines. Six different cDNA fragments of the PTK genes were isolated, and the sequence analysis showed that these represented cDNAs for TIE1, KDR, FMS, FGFR-4, JAK1 and HCK. Of these genes, the expression of TIE1, KDR, and FGFR-4 was studied in RCC tissue and cell lines by Northern blot analysis. We also investigated the expression of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptor FLT-1. In all the hypervascular RCC tissues, the amounts of mRNAs for KDR and FLT-1 were increased compared to adjacent normal tissues. The TIE1 and FGFR-4 genes were also overexpressed in most of the hypervascular RCC tissues, while no mRNA of KDR, FLT-1, or TIE1 could be detected in any of the four human RCC cell lines. The amounts of the VEGF and PlGF mRNAs were increased in hypervascular RCC tissues, while VEGF mRNA was detected in the four cell lines but PlGF mRNA was not. FGFR-4 mRNA was expressed in three of the four cell lines. These results suggest that KDR, FLT-1, PlGF and TIE1 mRNAs are present in the mesenchymal cells of RCC, while VEGF and FGFR-4 genes are expressed in RCC cells themselves in vivo.     

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.     

47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.     

Chromogranin A in the human gastrointestinal tract: an immunocytochemical study with region-specific antibodies.

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411-424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 17-38 and 176-195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.     

Heterogeneity of polyadenylation site of mRNA coding for human serum albumin

Human fetal liver cDNA was cloned in pBR322 vector by dG:dC-tailing method. The cDNA library was screened for liver-specific clones by means of differential hybridization. Human fetal liver and human kidney cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand recombinants analysed. These clones represent cDNAs corresponding abundant mRNAs. Eighteen clones were identified as encoding serum albumin. Two different mRNA polyadenylation sites were found in four sequenced plasmids. Cleavage/polyadenylation site in two plasmids, pHA1 and pHA12, is situated fifteen nucleotides downstream the AATAAA signal; in two other plasmids, pHA8 and pHA25, this site is ten nucleotides downstream the same signal.