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Biomedical subjects

K Zheng

Publications and source records attributed to K Zheng.

33 records · Page 2Linked to original sources
PubMed1996

Coordinate gene expression of the alpha3, alpha4, and alpha5 chains of collagen type IV. Evidence from a canine model of X-linked nephritis with a COL4A5 gene mutation.

Canine X-linked hereditary nephritis is an animal model for human X-linked hereditary nephritis with a premature stop codon in the alpha5(IV) gene of collagen type IV. We used this model to examine the other alpha(IV) chains at the mRNA and protein level in the kidney, since in human X-linked hereditary nephritis, the alpha3(IV) and alpha4(IV) chains are often absent from the glomerular basement membrane, although both are encoded by autosomal genes. cDNA probes for the alpha1(IV)-alpha6(IV) chains were generated from normal dog kidney using the polymerase chain reaction. Sequences were >/=88% identical at the DNA level and >/=92% identical at the protein level to the respective human alpha(IV) chains. By Northern analysis, transcripts for the alpha1(IV), alpha2(IV), and alpha6(IV) chains were detected at comparable levels in both normal and affected male dog kidney RNA. As previously shown, the transcript for the alpha5(IV) chain was reduced to approximately 10% of normal. Unexpectedly, the alpha3(IV) and alpha4(IV) transcripts were both decreased >/=77% in affected male dog kidney, suggesting a mechanism coordinating the expression of these three basement membrane components. The NC1 domain of collagen type IV isolated from normal dog glomeruli was positive for the alpha3(IV), alpha4(IV), and alpha5(IV) chains by Western blotting. In contrast, in the NC1 domain isolated from affected dog glomeruli, these three chains were not detectable, except for a trace of alpha3(IV) dimer. In X-linked hereditary nephritis, the absence of the alpha3(IV) and alpha4(IV) chains from glomerular basement membrane may reflect factors acting at the transcriptional and/or translational level in addition to the protein assembly level.

Amino Acid Sequence↗
PubMed1996

Capillary liquid chromatography coupled with an ion trap storage/reflectron time-of-flight mass spectrometer for structural confirmation of three recombinant protein isoforms.

Packed-capillary high-performance liquid chromatography (HPLC) was successfully coupled with an ion trap storage/reflectron time-of-flight mass spectrometer (LC/IT/reTOFMS) through an electrospray ionization interface for protein structural elucidation. Using the total-ion storage capabilities of the trap over a broad mass range and the high sensitivity from the packed capillary column with i.d. as small as 250 microns, high sensitivity peptide mapping in the low picomole range was demonstrated for the structural confirmation of three recombinant human nucleoside diphosphate kinase isoforms (NDPK, E.C. 2.7.4.6). A strategy combining chemical/enzymatic digestions as well as collisionally-induced dissociation (CID) in the electrospray source was successfully employed to infer the minor primary structural differences among the three recombinant proteins. This high sensitivity was achieved while also maintaining a resolution of nearly 1500 for mass identification using the capabilities of the IT/reTOF device. A point mutation of serine 120 to glycine was verified between the wild-type NDPK A and its mutant (delta m = 30 u) by both selected-ion monitoring and ion-source CID of the protein fragment containing the mutation site. For the structural confirmation of the sequence of NDPK A and B (88% homology), two sets of chemical/proteolytic digests were generated independently and followed by LC/MS analysis of the molecular weight of each protein-generated fragment. The complementary information from the two chromatographic analyses allowed for sequence verification of the two protein isoforms. The experiments clearly demonstrated that the high concentration sensitivity of the capillary high-performance liquid chromatographic separation together with the advantages of the IT/reTOF mass spectrometer could provide a low-cost, high-performance facility for protein analysis.

Amino Acid Sequence↗
PubMed1996

Determination of bacterial protein profiles by matrix-assisted laser desorption/ionization mass spectrometry with high-performance liquid chromatography.

A rapid method for profiling bacterial and cellular proteins has been developed using a combination of capillary high-performance liquid chromatography separation followed by (MALDI-MS) matrix-assisted laser desorption/ionization mass spectrometric analysis. In this method, bacteria are sonicated, the cell walls broken, and the water-soluble proteins precipitated for analysis. The proteins are separated by capillary liquid chromatography and detected on-line by a UV absorption detector. The eluents are then collected for off-line analysis by MALDI-MS. Using this method, it is demonstrated that bacteria can be discriminated based upon their protein profiles to the species level with only pmol level detection of proteins. It has also proved to be a fast and accurate means for monitoring the expression of Hsp27 in an insect cell system.

Animals↗
PubMed1995

Expression and chromosomal localization of cDNA clones from an enriched human retinal pigment epithelial (RPE) cell line library: identification of two RPE-specific genes.

We have previously constructed an enriched cDNA library from a human retinal pigment epithelium (RPE) cell line and generated expressed sequence tags (ESTs) from novel clones. Here, we report the analysis of expression of 14 cDNAs and identify two clones, AA1 and AA28, that appear to be specifically expressed in RPE but not in any other tissue tested. We have also localized 15 novel cDNAs (including the two RPE-specific cDNAs) to human chromosomes using in situ hybridization or in conjunction with somatic cell hybrid analysis. The cDNAs were mapped to the following chromosomal regions: 1p35-->p33, 1q41-->q42 (two clones), 3q11.2-->q13.1, 3q24-->q25, 4q13-->q21, 6q22-->q23, 7q34-->q36, 10q23-->q24, 11q23-->q24, 15q25-->q26, 19p13.3, 20p13, 21q11.2-->q21, and 21q22.2-->q22.3. The genetic and functional analysis of the two RPE-specific genes should contribute to a better understanding of RPE function. Chromosomal localization of RPE cDNAs will be valuable in identifying candidate genes for inherited diseases involving RPE dysfunction and aid in establishing the expression map of the human genome.

Blotting, Northern↗
PubMed1994

Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV.

Many families with X-chromosome linked hereditary nephritis (HN) have mutations in the gene on the X chromosome that codes for the alpha 5 chain of collagen type IV. Canine X-linked HN is an animal model for human X-linked HN. To study the alpha 5(IV) gene in this model, we used the nucleotide sequence published for the human alpha 5(IV) cDNA to construct sets of primers covering approximately 95% of the complete cDNA. cDNA from both affected and normal dog kidneys was amplified by PCR in nine overlapping regions. The nucleotide sequence encoding the noncollagenous domain NC1 hybridized to the human X chromosome and was 93% identical at the DNA level and 97% identical at the protein level to the human alpha 5(IV) NC1 domain, confirming that the canine alpha 5(IV) cDNA had been amplified. Sequence analysis of the alpha 5(IV) cDNA detected a single nucleotide substitution, G-->T, in affected dogs, changing a codon for a conserved glycine residue (GGA) to a stop codon (TGA). When genomic DNA was amplified, the same abnormality was found in exon 35. Using the canine NC1 domain cDNA as a probe for Northern analysis, two transcripts of approximately 8.6 kb and approximately 6.7 kb were identified in both normal and affected male dog kidney RNA. However, the abundance of both transcripts was decreased by a factor of approximately 10 in the affected dog. These results establish at the molecular level that canine X-linked HN is a model for human X-linked HN. This model provides an opportunity to determine the efficacy of new therapies and to investigate the role of the alpha 5(IV) chain in type IV collagen assembly.

Amino Acid Sequence↗
PubMed1992

Assignment of the human glycogen debrancher gene to chromosome 1p21.

Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but approximately 15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1p21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein.

Animals↗
PubMed1992

Colocalization of the genes for the alpha 3(IV) and alpha 4(IV) chains of type IV collagen to chromosome 2 bands q35-q37.

Each type of basement membrane in man contains between two and five genetically distinct type IV collagens: alpha 1(IV)-alpha 5(IV). Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been isolated. We have recently isolated partial cDNAs for the fifth member of the family, designated alpha 4(IV). On the basis of comparison of the deduced peptide sequences of all five chains, the type IV collagens can be divided into two families: alpha 1-like, comprising alpha 1(IV), alpha 3(IV), and alpha 5(IV); and alpha 2-like, comprising alpha 2(IV) and alpha 4(IV). Genes encoding the alpha 1(IV) and alpha 2(IV) chains (COL4A1 and COL4A2) both map to human chromosome 13q34 and have been shown to be transcribed from opposite DNA strands using a common bidirectional promoter that allows coordinate regulation of the two chains. Indeed, these two chains are commonly found together in basement membrane and form [alpha 1]2.[alpha 2] heterotrimers. Whereas alpha 1(IV) and alpha 2(IV) have been found in all basement membranes studied hitherto, it has been shown that alpha 3(IV) and alpha 4(IV) are found in only a subset of basement membranes. In basement membranes where either of these molecules is present, however, they are found together. In view of this relationship and the structural similarities between alpha 1(IV) and alpha 3(IV) and between alpha 2(IV) and alpha 4(IV), we hypothesized that COL4A3 and COL4A4, the genes encoding alpha 3(IV) and alpha 4(IV), respectively, have a genomic organization similar to that of COL4A1 and COL4A2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗
PubMed1992

Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter.

We have screened a human substantia nigra cDNA library with probes derived from the rat dopamine transporter. A 3.5-kilobase cDNA clone was isolated and its corresponding gene was located on the distal end of chromosome 5 (5p15.3). This human clone codes for a 620-amino acid protein with a calculated molecular weight of 68,517. Hydropathicity analysis suggests the presence of 12 putative transmembrane domains, a characteristic feature of sodium-dependent neurotransmitter carriers. The rat and the human dopamine transporters are 92% homologous. When permanently expressed in mouse fibroblast Ltk- cells, the human clone is able to induce a saturable, time- and sodium-dependent, dopamine uptake. This transport is blocked by psychostimulant drugs (cocaine, l- and d-amphetamine, and phenyclidine), neurotoxins (6-hydroxydopamine and N-methyl-4-phenylpyridine (MPP))+), neurotransmitters (epinephrine, norepinephrine, gamma-aminobutyric acid, and serotonin), antidepressants (amitriptyline, bupropion, desipramine, mazindol, nomifensine, and nortriptyline), and various uptake inhibitors (mazindol, GBR 12783, GBR 12909, and amfonelic acid). The rank orders of the Ki values of these substances at the human and the rat dopamine transporters are highly correlated (r = 0.998). The cloning of DNA human dopamine transporter gene has allowed establishment of a cell line stably expressing the human dopamine transporter and, for the first time, an extensive characterization of its pharmacology. Furthermore, these newly developed tools will help in the study of the regulation of dopamine transport in humans and in the clarification of the potential role of the dopamine transporter in a variety of disease states.

Amino Acid Sequence↗
PubMed1991

Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the alpha subunit.

The human serine/threonine protein casein kinase II (CK II) contains two distinct catalytic subunits, alpha and alpha 1, which are encoded by different genes. A combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization have localized the human CK II-alpha DNA sequence to two loci: 11p15.5-p15.4 and 20p13. In contrast, the CK II-alpha' gene has been mapped to chromosome 16 by somatic cell hybrid analysis. Taken together with our previous assignment of the CK II regulatory beta-subunit gene to 6p12-p21, these results indicate that although the products of these genes form a single biological complex, they are encoded on different human chromosomes. Further analysis should determine whether both loci of CK II-alpha are functional, or perhaps one of the two constitutes a pseudogene.

Blotting, Southern↗
PubMed1987

Complete nucleotide sequence of reovirus L2 gene and deduced amino acid sequence of viral mRNA guanylyltransferase.

Reovirus mRNAs synthesized by the virion-associated RNA polymerase contain a 5'-terminal cap that is added to nascent transcripts by polypeptide lambda 2, a structural component of virions encoded by double-stranded RNA genome segment L2. The complete, 3916-nucleotide sequence of a full-length reovirus type 3 L2 DNA clone was determined by the dideoxy chain terminator method. The sequence has a single long open reading frame extending from the second A-T-G at nucleotide 14 to a termination codon at position 3881. On this basis, the 1289-amino acid sequence of polypeptide lambda 2, the reovirus mRNA guanylyltransferase, was deduced and compared to other GTP-binding proteins. Two different, lysine-containing lambda 2 peptide sequences closely resemble predicted amino acid stretches in vaccinia virus guanylyltransferase and potentially form part of active sites in the viral mRNA capping enzymes.

Amino Acid Sequence↗
PubMed1987

A conserved nucleotide sequence at the sites of developmentally regulated chromosomal breakage in Tetrahymena.

Chromosomal breakage occurs at hundreds of specific sites in Tetrahymena, including the two ends of the unique ribosomal RNA genes, during the development of the somatic macronucleus. We have identified a 15-nucleotide sequence that occurs widely in the germinal micronuclear genome and is associated exclusively with chromosomal breakage sites. When copies of this sequence were cloned and analyzed, they were found in all cases to be located at or very near sites of breakage. This general rule is further supported by the observation that in a different site in which a single nucleotide substitution is found within this sequence, no chromosomal breakage occurs. The complete sequence structure of one of the breakage junctions has also been determined in both the germinal DNA and the two somatic DNA termini. This structure reveals that the 15-nucleotide conserved sequence is located within a 54-nucleotide region that is removed following chromosomal breakage.

Animals↗