Intravenous lipid dose and incidence of bacteremia and fungemia in patients undergoing bone marrow transplantation

To identify genes specifically expressed in taste tissues, we constructed a subtraction cDNA library of epithelium of rat circumvallate and foliate papillae and carried out differential screening of this library. Dot blot analysis showed 46 out of 88 clones obtained by this method to be expressed in the epithelium of papillae. The cDNA inserts in these clones were sequenced and analyzed for similarity to entries the GenBank database. About 54.3% of the clones were known sequences, including the sequences of ebnerin, cytokeratin 18, and Na+,K+-ATPase, that were shown by in situ hybridization to be expressed in the circumvallate papillae. About 41.3% of the papillae-specific clones had no significant similarity to known sequences and are candidates for novel taste bud-specific marker genes.     

Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis

Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.     

Toward a cDNA map of the human genome

Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosome band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for teh rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to map 41 cDNAs with an average insert size of <2 kb to single human chromosome bands. The result provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular signal-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphatase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (2-5 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases.     

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.     

Psychopathology, treatment completion and 5 years outcome. A prospective study of drug abusers

The strategy of isolating the band-specific expression fragments from a probe pool generated by human chromosome microdissection was reported. A chromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1-32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but can't hybridize to 17q11-12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences, and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.     

Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains

Using cDNA subtraction and differential hybridization techniques, a cDNA library derived from normal quail embryo fibroblasts was screened for clones corresponding to genes whose expression was suppressed in v-myc-transformed, as compared with normal, quail embryo fibroblasts. One of the isolated cDNA clones corresponded to a 0.9-kb mRNA that was present in normal quail and chicken embryo fibroblasts, but was virtually absent from all transformed avian cells tested: quail embryo fibroblasts transformed by the v-myc, v-myc/v-mil or v-src oncogenes, cells derived from a methylcholanthrene-induced quail fibrosarcoma or v-myc-transformed chicken macrophages. Nucleotide sequence analysis of the original and supplementary cDNA clones indicated that the corresponding gene encodes a 194 amino acid cysteine-rich protein (M(r) 20,911). A database search revealed that the gene is the avian homolog of a human primary response gene (crp) of unknown function. Both the quail and human CRP proteins contain two copies of a cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity that was previously identified in several proteins with presumed regulatory functions essential for cell growth or differentiation.     

A systematic molecular genetic approach to study mammalian germline development

It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the beta-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for beta-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process.     

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.     

Gene expression of lipid binding protein transferred the ability of specific attachment of hemopoietic cells to non-supportive stromal cell line, MS-K

In order to identify the cDNAs responsible for hemopoietic supportive activity, expression of mRNAs in hemopoietic supportive bone marrow stromal cells (MS-5) and non-supportive stromal cells (MS-K) were compared by the cDNA subtraction method. A subtracted MS-5-cDNA library, which contains cDNA clones corresponding to mRNAs present in MS-5 cells but not in MS-K cells, was constructed. Screening of subtracted MS-5-cDNA library resulted in the isolation of some clones. Two of them, lipid binding protein (LBP) and haptoglobin (Hp), were expressed specifically in MS-5 cells but not in MS-K cells. The genes of LBP and Hp were subcloned into mammalian expression vector and transfected into hemopoietic non-supportive stromal cells line, MS-K. Then LBP-expressing stable transformants (MS-K-LBP) and Hp-expressing transformants (MS-K-Hp) were cloned. A rosette formation assay was carried out to investigate whether or not the LBP and Hp cause MS-K cells to adhere to hemopoietic cells. MS-K-LBP formed rosettes with hemopoietic cells as MS-5 cells, although the MS-K-Hp and normal MS-K cells did not form rosettes. These data indicate that LBP expressed in hemopoietic supportive stromal cells is partly responsible for the adhesion of hemopoietic stem cells to stromal cells.     

Cloning in silico

Cellulose is a major component of plant cell walls, but identification of the enzymes that synthesize it has proven difficult. Now, however, several candidate proteins with sequence homology to bacterial cellulose synthases have been identified by partial sequencing of anonymous cDNA clones from cotton fibers.     

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Objective To construct a morphine tolerance model in primarily cultured striatal neurons,and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours).To check reliability of the cell culture model,RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression.The subtracted clones were prescreened by PCR.The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis.And the expression levels of genes of interest were confirmed by RT-PCR.Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.8S±1.98) compared with normal striatal neurons (28.43±1.46,P<0.01).Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis.And the 36 clones showed homology with 19 known genes and 2 novel genes.The expression of 2 novel genes,mitochondrial carrier homolog 1 (Mtchl; 96.81±2.04 vs.44.20±1.31,P<0.01) and thymoma viral proto-oncogene 1 (Aktl; 122.10±2.17 vs.50.11±2.01,P<0.01),showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons.Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed.Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.     

Outpatient inotropic therapy in heart transplant candidates: should its use influence waiting list priority status?

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20-30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an approximately 20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF052831-AF052833.]     

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.     

Glutaredoxin is a direct target of oncogenic jun

We have analysed differential gene expression in v-jun-transformed chicken embryo fibroblasts (CEF) compared to normal CEF by using the directional tag PCR subtraction method. From a first generation of putative Jun targets four clones were selected for study; they are upregulated in jun-transformed cells. Three of these clones showed homology to known genes: glutaredoxin, growth associated protein (GAP)-43/neuromodulin, and phenobarbital-induced cytochrome P450. The expression of these genes was analysed in fibroblasts transformed by various oncogenes. Expression of the glutaredoxin mRNA could be induced by a Jun-estrogen receptor chimaera in the absence of de novo protein biosynthesis. Based on this observation we conclude that glutaredoxin is a direct target of v-Jun.