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Biomedical subjects

Z Kelman

Publications and source records attributed to Z Kelman.

13 recordsLinked to original sources
PubMed1995

Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitope.

Escherichia coli expression vectors, based on the pET system, were constructed to allow fusion of a protein kinase (PK) recognition motif, a hemagglutinin (HG) epitope-tag and a His6-tag at the N-terminal portion of a protein of interest. The fusion proteins, that result from expression using these vectors, can be phosphorylated in vitro using cAMP-dependent PK, immunoprecipitated using monoclonal antibody against the HG-epitope, and can be rapidly purified using a Ni2+ column.

Amino Acid Sequence↗
PubMed1995

Protein structure. Why have six-fold symmetry?

Why do proteins proteins that encircle DNA have six-fold symmetry? One important factor may be the economy in protein mass with which DNA can be encircled by six globular subunits arranged in a ring.

Animals↗
PubMed1995

Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps.

The remarkable processivity of cellular replicative DNA polymerases derive their tight grip to DNA from a ring-shaped protein that encircles DNA and tethers the polymerase to the chromosome. The crystal structures of prototypical 'sliding clamps' of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for encircling DNA. Although beta is a dimer and PCNA is a trimer, their structures are nearly superimposable. Even though they are not hexamers, the sliding clamps have a pseudo 6-fold symmetry resulting from three globular domains comprising each beta monomer and two domains comprising each PCNA monomer. These domains have the same chain fold and are nearly identical in three-dimensions. The amino acid sequences of 11 beta and 13 PCNA proteins from different organisms have been aligned and studied to gain further insight into the relation between the structure and function of these sliding clamps. Furthermore, a putative embryonic form of PCNA is the size of beta and thus may encircle DNA as a dimer like the prokaryotic clamps.

Amino Acid Sequence↗
PubMed1995

DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine.

DNA polymerase III holoenzyme contains two DNA polymerases embedded in a particle with 9 other subunits. This multisubunit DNA polymerase is the Eschericia coli chromosomal replicase, and it has several special features that distinguish it as a replicating machine. For example, one of its subunits is a circular protein that slides along DNA while clamping the rest of the machinery to the template. Other subunits act together as a matchmaker to assemble the ring onto DNA. Overall, E. coli DNA polymerase III holoenzyme is very similar in both structure and function to the chromosomal replicases of eukaryotes, from yeast all the way up to humans. This review summarizes our present knowledge about the function of the 10 subunits of this replicating machine and how they coordinate their actions for smooth duplication of chromosomes.

Adenosine Triphosphate↗
PubMed1994

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.

CDC2-CDC28 Kinases↗
PubMed1994

DNA replication: enzymology and mechanisms.

Research into the enzymology of DNA replication has seen a multitude of highly significant advances during the past year, in both prokaryotic and eukaryotic systems. The scope of this article is limited to chromosomal replicases and origins of initiation. The multiprotein chromosomal replicases of prokaryotes and eukaryotes appear to be strikingly similar in structure and function, although future work may reveal their differences. Recent developments, elaborating the activation of origins in several systems, have begun to uncover mechanisms of regulation. The enzymology of eukaryotic origins has, until now, been limited to viral systems, but over the past few years, enzymology has caught a grip on the cellular origins of yeast.

DNA Replication↗
PubMed1993

The murine vik gene (chromosome 9) encodes a putative receptor with unique protein kinase motifs.

Receptor tyrosine kinases are involved in the regulation of cell growth and may play a central role in embryonic development. We recently developed a polymerase chain reaction (PCR)-based gene cloning procedure that allows selective isolation of genes that encode novel transmembrane tyrosine kinases in tissues of embryonic origin. By employing this protocol on mRNA from a 12.5 day post-coitum mouse placenta, we identified a gene for a putative receptor protein kinase. The deduced amino acid sequence predicts the existence of an approximately 200 amino acid long extracellular domain that shows no similarity to known proteins. The cytoplasmic portion contains a core sequence that is structurally homologous to the tyrosine kinase family. However, a few highly conserved short blocks of sequences, shared by all protein kinases, display variations in the isolated gene. These include the glycine-rich block at the nucleotide-binding cleft and the Asp-Phe-Gly triplet at the substrate recognition site. On the basis of these variations, we named the gene vik for variant in the kinase. Northern analysis revealed two widely expressed transcripts of vik with molecular weights of 3 and 2.5 kb. Chromosomal mapping using restriction fragment length polymorphism localized the gene to murine chromosome 9. The unique structural landmarks of vik at both the extracellular and the cytoplasmic domains suggest novel ligand as well as substrate specificity of the presumed receptor.

Amino Acid Sequence↗
PubMed1992

Signal transduction by the neu/erbB-2 receptor: a potential target for anti-tumor therapy.

The neu/erbB-2 protooncogene encodes a transmembrane tyrosine kinase homologous to receptors for polypeptide growth factors. The oncogenic potential of the presumed receptor is released through multiple genetic mechanisms including a point mutation, truncation of non-catalytic sequences and overexpression. The latter mechanism appears to be relevant to human cancers as elevated expression of the neu/erbB-2 gene is frequently observed in solid tumors of various adenocarcinomas. It is therefore conceivable that strategies aimed at the biochemical mechanism of action of the neu/erbB-2 tyrosine kinase may contribute to the treatment of certain human cancers. To this aim we undertook a multiple research approach consisting of the following directions: (i) The neu/erbB-2 ligand--a systematic screening of potential biological sources of the hypothetical hormone molecule, that presumably binds to the neu/erbB-2 protein, resulted in detection of a candidate activity in the medium of certain cultured transformed cells. Partial purification indicated that the factor is a 30-35 kDa glycoprotein. Further studies revealed several biochemical characteristics of the factor that may be helpful for complete purification and structural analysis of this novel hormone. (ii) Signal transduction by neu/erbB-2--using a chimeric receptor approach and various mutants we found that all the oncogenic forms of the neu/erbB-2 are constitutively coupled, both physically and functionally, to a multi-protein complex of signaling molecules. The latter includes the phosphatidylinositol-specific phospholipase C gamma and a phosphatidylinositol kinase. Thus, the metabolism of inositol lipids is probably a major biochemical pathway utilized by the neu/erbB-2 tyrosine kinase. (iii) Tumor inhibitory antibodies--we generated a panel of monoclonal antibodies to the presumed receptor. Surprisingly, some antibodies almost completely inhibited the growth of tumor cells in athymic mice, whereas one antibody significantly accelerated the rate of tumor growth in animals. Interestingly, the inhibitory antibodies conferred a mature phenotype to cultured breast cancer cells, implicating terminal differentiation in tumor retardation.

Amino Acid Sequence↗
PubMed1991

PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors.

Transmembrane tyrosine kinases are involved in the control of cell growth and differentiation by extracellular signals. To enable identification of new receptor tyrosine kinases we developed a method that selectively amplifies segments of receptor genes. The method is based on a combination of polymerase chain reaction (PCR) and hybridization screening and it employs three oligonucleotide primers derived from conserved domains of receptor tyrosine kinases. It yields amplification of receptors' genes and appears to ignore cytoplasmic tyrosine kinases. When applied to RNA from 12.5 days post coitum mouse placenta, this methodology resulted in the detection of several putative or established receptors. Molecular cloning of one of these genes, which is identical to the partially characterized bek gene, identified a transmembrane tyrosine kinase with three immunoglobulin-like domains in the extracellular portion, and a cytoplasmic tyrosine kinase sequence. The isolated cDNA shows remarkable homology to the murine flg gene that encodes a receptor for fibroblast growth factors. Indeed, an antibody directed to the carboxy terminus of the deduced bek protein specifically recognized a receptor for acidic and basic fibroblast growth factors in murine hepatoma cells. We therefore expect that the methodology we developed will enable the study of new receptors in hardly accessible biological systems such as early mammalian embryos or stem cells.

Amino Acid Sequence↗
PubMed1990

Chromosomal mapping of the murine c-abl proto-oncogene by in situ hybridization.

Deletion and rearrangement of chromosome 2 were shown to be major cytogenetic characteristics of radiation-induced murine myeloid leukemias. Analysis of the localization of the murine protooncogene c-abl, previously assigned by Goff et al. to chromosome 2, was done using the in situ hybridization method. The c-abl was located close to the centromere, within bands 2A-2B. This site does not correspond to the common characteristic deleted segments (2C-2D) predominantly observed in radiation induced murine myeloid leukemias.

Animals↗
PubMed1989

Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia.

Molecular structural analysis of the p53 gene in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) indicates a significant incidence of gene rearrangements in patients at either accelerated phase or blastic crisis. Southern blot analysis of genomic DNA hybridizing with either genomic or cDNA p53 specific probes indicated that 30% of the CML patients at blastic crisis phase exhibited rearrangements, mostly mapping downstream to the first non-coding exon. This is compatible with the observation that the progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. Therefore, we suggest that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene.

Blast Crisis↗