Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.     

对比分析黑斑息肉综合征合并大肠癌组织肿瘤基因

Objective: The aim of the study was to screen the differentially expressed genes of Peutz-Jeghers syndrome (PJS) and colorectal carcinoma (CRC). Methods: This study used cDNA microarray to comparatively analyze the gene expression profiles of 4 cases of PJS combined with colorectal adenocarcinoma vs. normal mucosae. The cDNA microarray contained 8064 human genes, and then using RT-PCR to test three genes of all. Results: The experimental data showed that fourteen genes were differentially expressed, which were up-regulated in PJS. Fifty-one genes expressions were altered in CRCs, of which 32 were up-regulated, as compared to the normal mucosae. In addition, 5 genes were similarly altered in both PJS and CRCs. RT-PCR analyses confirmed the cDNA microarray data for three of those genes: LATS2, APC and MADH4. Conclusion: LCN2, USP4, GRO3, HYAL1 and APC - these differentially expressed genes likely represent biomarkers for early detection of CRC and may be potential therapeutic targets.     

Characterization of alkaline phosphatase genes expressed in seminoma by cDNA cloning

Two members of a placental alkaline phosphatase (PLAP) family, PLAP and PLAP-like or germ cell alkaline phosphatase, are aberrantly expressed in tumors of ecotropic origin. To characterize alkaline phosphatase induced in seminoma, alkaline phosphatase cDNA clones were isolated from a cDNA library constructed from seminoma cells and characterized by nucleotide sequence determination. Thus, isolated cDNA clones were classified into two types, germ cell alkaline phosphatase (PLAP-like) and liver/bone/kidney-type alkaline phosphatase (L/B/K AP). These results suggest that other than the PLAP family members, the expression of L/B/K AP is enhanced in seminoma and can serve as a tumor marker in seminoma.     

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.     

Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.     

Carrot DNA-methyltransferase is encoded by two classes of genes with differing patterns of expression

There is a wide variety of renal tubular disorders affecting children. Understanding the pathophysiology of each disease assists in the recognition and treatment of these diseases, which can have profound effects on a child's growth and development. We review some of the more common tubular disorders found in children, emphasizing those conditions in which recent advances have been made in understanding of the specific genetic or physiological defect responsible: Fanconi's syndrome, cystinuria, renal tubular acidosis, and the hypokalemic metabolic alkaloses. Current recommendations are presented for diagnosing and treating these conditions.     

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.     

Isolation of differentially expressed genes upon immunoglobulin class switching by a subtractive hybridization method using uracil DNA glycosylase

Immunoglobulin class switch recombination enables B lymphocytes to sequentially express antibodies that have identical specificities but that differ in class and effector function. Although several cis elements required for class switch recombination have been identified, few trans -acting factors which are directly related to class switching have been found. Previously we have developed an efficient in vitro class switching system using a cell line, CH12F3-2. To clarify the molecular mechanism of class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L using the in vitro class switching system. For that purpose, an improved method for making subtracted cDNA libraries, using uracil DNA glycosylase, has been developed. This method can overcome a general problem of subtraction, that rare cDNAs are easily lost. This new subtraction method was applied to the CH12F3-2 switching system to isolate genes induced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encoding deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained. Their expression patterns in response to various combinations of stimuli and the time courses of the induction supported the usefulness of this method.     

A catalogue of genes in the cardiovascular system as identified by expressed sequence tags

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.     

Epithelial marker genes are expressed in cultured embryonic rat lung and in vivo with similar spatial and temporal patterns

Explants of embryonic lung are often used to characterize lung growth, bronchial tree pattern, and cell differentiation. Most investigators culture lungs for 3-7 days in defined media lacking, e.g., added growth factors or hormones. If growth and differentiation are comparable to that in vivo, these cultures show considerable promise for identifying developmental regulatory molecules and target genes, and for elucidating molecular responses. We used in situ hybridization and RT-PCR to compare times and sites of expression of mRNAs of six epithelial genes in cultured and uncultured fetal rat lungs. These genes, expressed in distal lung of adult rats, are surfactant proteins (SP) A, B, and C; LAR, a receptor-type tyrosine phosphatase; Clara cell secretory protein (CC10, CCSP); and T1alpha. SP-A, SF-B, LAR, and CC10 are expressed by both Clara and Type II cells in adult animals. SP-C and T1alpha are unique markers for Type II and Type I cells, respectively. SP-C, LAR, and T1alpha are expressed before the lung is explanted (Day 13.5); SP-A, -B, and CC10 mRNAs are first detected later. The onset of expression is similar in vivo and in vitro. Although the patterns of expression differ for each mRNA, their sites of expression in culture match those in vivo relative to the bronchial tree. The explanted embryonic lung appears to be an excellent experimental model.     

Molecular cloning and expression of cynomolgus monkey interleukin-2 cDNA by the recombinant baculovirus system

Mice inoculated at 5, 21 and 28 days of age with 10(6) or 10(7) Cryptosporidium parvum oocysts became infected but did not exhibit any clinical signs of disease. Specific IgA antibodies were detected in faecal extracts from all infected mice by an indirect immunofluorescent assay. These antibodies first appeared between 11 and 37 days post-infection (dpi) and persisted until the end of the experiment at 55 dpl. They appeared earlier in older mice than in newborn mice. Reduction and resolution of oocyst shedding was not directly related, however, to IgA antibody levels in infected mice. Reactive C. parvum antigens were identified by immunoblotting techniques using faecal and serum samples from infected mice. IgA copro-antibodies reacted specifically with two antigens of 26 and 33 kDa, which were also identified by IgG antibodies in mouse serum. The role of these antibodies in the resolution of infections and the subsequent protection against challenge is unknown.     

Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning

Pregnancy block, whereby recently mated female mice abort their pregnancies when exposed to novel (strange) males, was studied in house mice (Mus domesticus) differing in t-complex genotype; t-mutations are deleterious and +/t females avoid +/t males as mates. The results of Experiment 1, in which the genotype of the female, stud male, and strange male was systematically varied, showed that pregnancy block was most frequent when the strange male was +/+. Because this effect was not enhanced among +/t females when stud males were +/t, the results cannot clearly be explained by the hypothesis that pregnancy block is a manifestation of mate choice. Moreover, the "strange male" effect in Experiment 1 is unlikely to be a female response correlated with the risk of male infanticide, as +/+ and +/t males did not differ in their infanticidal tendencies (Experiments 2 & 3). Alternative hypotheses, including a modified version of the mate choice hypothesis, are discussed.