Biomedical subjects
Publications and source records attributed to T J Kelly.
Physicians and nurses need to be aware of the metabolic needs of the diabetic patient and incorporate those requirements into the plan of care during the perioperative period. With appropriate management, morbidity and mortality rates for diabetic patients who undergo surgery may approach those for the nondiabetic population.
Treatment of purified simian virus 40 large T antigen (LT) with protein phosphatase 2A stimulates LT-dependent DNA unwinding and replication (D. M. Virshup, M. G. Kauffman, and T. J. Kelly, EMBO J. 8: 3891-3898, 1989). The specificity of the catalytic subunit of protein phosphatase 2A toward LT was investigated by two-dimensional peptide mapping. Increasing amounts of phosphatase sequentially removed the phosphates from serine residues 120, 123, 677, and perhaps 679, residues which have been implicated in regulating the DNA-binding activity of LT.
The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.
Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.
The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.
Thymidine kinase (TK) is a nucleotide salvage pathway enzyme whose activity is highly dependent on the growth state and cell cycle phase of a cell. Cells in the resting or quiescent (G0) phase express very low levels of TK mRNA and protein. When quiescent cells are stimulated to enter the cell cycle by the addition of serum, TK mRNA, activity and polypeptide increase coordinately after about 10-15 h, at the beginning of S phase. When growth-independent heterologous promoters are substituted for the natural TK promoter, TK mRNA can be expressed in quiescent cells. Despite the presence of TK mRNA in such G0 cells, there is little expression of TK polypeptide; the normal increase in enzyme at S phase is observed following serum stimulation. Deletion of the introns and 3' untranslated sequences does not affect the expression of the TK gene in serum stimulation experiments. In contrast, deletion of the C-terminal 40 amino acids or fusion of a small segment of a beta-galactosidase to the C-terminus overcomes the block to expression of the TK polypeptide in G0 cells. These C-terminal alterations are the same as those which lead to constitutive expression of TK during the cell cycle of proliferating cells, suggesting that mechanisms which control the levels of TK in cycling cells may also operate in quiescent cells.
Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.
Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.
The prothoracic glands (PGs) of Lymantria dispar (day-5 female, last-stage larvae) produce both ecdysone and an ecdysteroid which has the same retention time on reverse-phase liquid chromatography (RPLC) as a known standard of 3-dehydroecdysone. The latter ecdysteroid can be converted by a heat-labile factor in extracts of post-diapause, pre-hatch L. dispar eggs to an ecdysteroid which has the same retention time on RPLC as ecdysone. Purified 3-dehydroecdysone, similarly treated with egg extract, also gives the same retention time on RPLC as ecdysone. Taken together, these data suggest that, like Manduca sexta, a major product of the PGs in L. dispar is 3-dehydroecdysone. Furthermore, these data suggest that L. dispar eggs, which contain mature embryos, possess ecdysteroid ketoreductase activity capable of converting 3-dehydroecdysone to ecdysone. This is the first report of ecdysteroid ketoreductase activity in embryonated eggs.
Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.
Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.
We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.
SV40 DNA replication in vivo is greatly stimulated by cis-acting transcriptional elements. We studied a model viral chromosome containing a single binding site for the cellular transcriptional activator, nuclear factor I (NF-I/CTF), located adjacent to the replication origin. The presence of the NF-I recognition site increased replication efficiency over 20-fold in vivo. Purified NF-I had little effect on the replication efficiency in the standard SV40 cell-free system when the template was introduced as naked DNA. However, NF-I specifically prevented the repression of DNA replication that occurred when the template was preassembled into chromatin. Our data support a model in which the binding of a transcriptional activator perturbs the local distribution of nucleosomes, thereby increasing the accessibility of the origin region.
Previously we isolated and characterized a differentially expressed gene from mouse 3T3-L1 preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300). Genomic Southern blot analysis indicated the existence of another closely related gene. Here we report the isolation and characterization of this gene and the corresponding cDNA which encode a second stearoyl-CoA desaturase, SCD2, 3T3-L1 adipocytes. SCD2 cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with greater than 87% amino acid sequence identity to SCD1. RNase protection analysis reveals a 10-fold increase in the expression of SCD2 mRNA during 3T3-L1 preadipocyte differentiation. SCD2 mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCD1 mRNA expression differs markedly from that of SCD2 mRNA being absent from brain, constitutive in adipose tissue, and subject to negative control in liver by feeding a diet containing unsaturated fatty acids. The SCD2 gene spans approximately 15 kilobase pairs and consists of six exons and five introns, with intron/exon junctions similar to those of SCD1. As determined by primer extension analysis the start site of transcription maps 300 nucleotides upstream of the initiator methionine codon. Unlike the SCD1 gene, SCD2 lacks a typical "TATA" box in the 5'-flanking region, but has two "CCAAT" boxes at positions -90 and -135 relative to the transcription initiation site. The SCD2 promoter contains a 140-base pair sequence (located between nucleotides -54 and -201) which possesses 77% sequence identity to a region (located between nucleotides -472 and -325) in the SCD1 promoter. There is a GC-rich sequence in the SCD2 promoter (at nucleotide -175) similar to the binding site for the nuclear transcription factor Sp1 as well as an element with homology to the core consensus sequence for the glucocorticoid regulatory element position -500 and a potential CCAAT box/enhance binding protein sequence at position -540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.
Human CTF/NF-I consists of a family of CCAAT box binding proteins that activate both transcription and DNA replication. Analysis of cDNA mutants expressed in E. coli and Drosophila cells reveals that the N-terminal portion of CTF-1 is sufficient for site-specific DNA recognition, protein dimerization, and adenovirus replication. In contrast, transcriptional activation requires an additional C-terminal domain. Furthermore, this transcription domain efficiently activates a heterologous promoter, such as SV40, when fused to the DNA binding domain of Sp1. The CTF C-terminal region consists of an unusual type of transcriptional activation domain containing approximately 25% proline residues. We propose that this proline-rich domain represents a novel class of activators which are distinct from those containing either acidic or glutamine-rich activation motifs. This indicates that transcriptional activation is likely to be mediated by several different mechanisms. In addition, these results suggest that the interactions, and consequently the mechanisms, governing transcriptional activation by CTF are distinct from those mediating DNA replication.
Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.
The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.
During differentiation of 3T3-L1 preadipocytes into adipocytes, expression of the gene encoding adipocyte 422(aP2) protein is activated. We have shown that the first 248 base pairs of the 422(aP2) gene promoter (which lacks a consensus cAMP response element) are sufficient to confer inducibility of a reporter gene by cAMP in preadipocytes. We now demonstrate by deletion analysis that this DNA segment contains overlapping negative and positive regulatory elements. The positive regulatory element contains a consensus activator protein 1 (AP-1) binding sequence. The effect of the negative regulatory element is observed in preadipocytes but not in fully differentiated adipocytes, suggesting that it is an important component of the regulatory mechanism governing expression of the 422(aP2) gene during differentiation. cAMP activates the 422(aP2) promoter in confluent preadipocytes but not in proliferating preadipocytes or fully differentiated adipocytes. The stimulatory effect of cAMP is abolished by deletions that enter the negative element, suggesting that cAMP increases expression by relieving the inhibitory effect of the negative regulatory element.