Landscape phage ligands for PC3 prostate carcinoma cells.

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display technology with its high throughput capacity for the analysis of targeting ligands possessing specific binding properties represents a very attractive tool in the quest for molecular ligands. Also, current phage nanobiotechnology concepts allow the use of intact phage particles and isolated phage coat proteins per se as components of nanomedicines. Herein, we describe the use of two landscape phage libraries to obtain phage ligands against PC3 prostate carcinoma cells. Following a very stringent selection scheme, we were able to identify three phage ligands, bearing the fusion peptides, DTDSHVNL, DTPYDLTG and DVVYALSDD that demonstrated specificity and selectivity to PC3 cells based on target-association assays, microscopy and flow cytometry. The phage ligands and their fusion coat proteins can be used as navigating modules in both therapeutic and diagnostic approaches to prostate carcinoma.

Expression and sequence of canine SIRT2 and p53 genes in canine mammary tumour cells - effects on downstream targets Wip1 and p21/Cip1.

We have characterized gene dysfunction in a cellular model of spontaneous canine mammary cancer by investigating specific gene defects in SIRT2 and p53 genes for comparative studies among canine tumour-derived cell lines. These genes and their downstream targets are involved in regulating gene silencing, cell cycle progression and prevention of senescence and apoptosis. Canine SIR2 reverse transcriptase-polymerase chain reaction amplicons were most homologous to human SIRT2 and revealed detectable transcripts in all cell lines. Canine SIRT2 contained non-conserved amino acid substitutions, representing mutations or allelic differences and interspecies differences. Sequence differences between individuals in p53 and SIRT2 were found in two cell lines including a stop codon in p53 and substitutions of conserved cysteine residues in the Zn(2+)-binding motif in SIRT2. Mutations in SIRT2 were coincident with expression of the p53 modulator, Wip1; a failure to activate p21/Cip1 and extended G2/M phase. A third cell line appeared to function normally in these two pathways and likely possesses other defects in proliferation-control genes. This data identify potentially important defects in pathways regulated by p53 and SIRT2 that modulate cell proliferation and integrate development, apoptosis and proliferative lifespan. These genes offer promising therapeutic targets, contributing to the transformed/immortalized phenotype in spontaneous canine mammary cancer.

Characterization of the enhancer-like okadaic acid response element region of the cyclin-dependent kinase 1 (p34cdc2) promoter.

Expression of the cdk1 (p34cdc2) gene is enhanced 5-10 fold as cells re-enter the cell cycle from quiescence in response to serum-refeeding or following exposure to the protein phosphatase 1/2A inhibitor okadaic acid. Transient transfection analysis of nested deletions of the human cdk1 promoter identified regions that confer sensitivity to okadaic acid on a CAT-reporter gene. Putative okadaic acid response elements (OARE) were located between nt -942 to -763 (Site I) and nt -416 to -186 (Site II) before transcription start. The Site I element has enhancer-like characteristics as activity is independent of sequence orientation. Mobility shift analysis of Site I revealed the presence of 2 high molecular weight complexes, one of which was enhanced in the presence of okadaic acid-treated cell extracts. Site I contained several sequence motifs with conserved homology to heat shock response element core sequences and homeobox protein binding sites. Site II contained a myb-binding site, a G1/S phase enhancer, and 2 retinoblastoma response elements flanking an E2F binding site. Enhancement of cdk1 expression appears dependent on 2 nonhomologous okadaic acid-sensitive promoter regions.

Expression kinetics and mapping of the thymidine kinase transcript and an immediate-early transcript from channel catfish virus.

Three transcripts from the terminal repeat of the channel catfish virus (CCV; also known as ictalurid herpesvirus 1) genome were mapped by S1 nuclease and primer extension analyses as well as by cDNA sequencing. These transcripts, TR3, TR5/6, and TR6, are encoded by open reading frame (ORF) 3, ORFs 5 and 6, and ORF 6, respectively, and correspond to those previously identified by sequence analysis (A. J. Davison, Virology 186:9-14, 1992). ORF 5 has previously been determined to encode thymidine kinase, but ORF 3 and ORF 6 encode proteins of unknown function. Although all three transcripts accumulate to high levels in cells infected in the presence of cycloheximide, kinetic analysis demonstrates that TR5/6 and TR6 are either early or late transcripts that leak through the cycloheximide block. In addition, two transcripts from the terminal repeat of the CCV genome that were mapped previously and were thought to be immediate-early in character, TR8a/9 and TR9, exhibit kinetics characteristic of early or late transcripts. TR3 is an immediate-early transcript that appears to have a very short half-life. In the 3' untranslated region of TR3, there are three copies of an AU-rich element which has previously been shown to be involved in destabilization of the oncogene c-fos and granulocyte/macrophage colony-stimulating factor mRNAs. mRNA destabilization may represent another mechanism by which herpesviruses regulate the rapid switch in expression from immediate-early genes to early genes during the transition to the early phase of infection.

Interaction of the Rb tumor suppressor protein with the c-fos promoter in c-fos transfected cells overexpressing c-fos and Rb.

The Rb tumor suppressor protein is overexpressed in HeLa cell lines permanently transfected with a constitutively expressed c-fos gene. CP17-14 cell overexpression of Rb may be due to a balancing response to overexpression of the stimulatory effects of c-fos overexpression on transcription. The cis-acting retinoblastoma control element (RCE, -97 to -86 bp) in the human c-fos promoter is thought to allow regulation of c-fos by Rb. Gel-shift assays were performed with a 168 bp fragment encoding the c-fos RCE. Competition assays with increasing mass of unlabeled probe or dose-dependence assays using increasing mass of nuclear proteins, demonstrated sequence-specific complex formation. Indistinguishable complexes were formed between the c-fos RCE fragment in transfected cells, but at higher levels (> 50%), compared to proteins from parental cells. Supershift analysis utilizing epitope-specific Rb-monoclonal antibodies indicated the presence of Rb protein bound to the RCE-containing DNA fragment. In contrast, polyclonal anti-Rb antibodies enhanced the amounts of nuclear protein-DNA complexes detected but did not result in a supershift. These results suggested the presence of Rb and/or Rb-like peptides involved in complex formation and the presence of multiple variants of RCE-binding complexes in response to c-fos over-expression.

Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines.

OBJECTIVE: To determine, for canine mammary tumors, whether malignancy, with or without local invasion or regional metastasis, was associated with overexpression of the oncogene c-erbB-2. DESIGN: c-erbB-2 expression was measured in canine mammary tumor-derived cell lines and in mammary tumor tissues from clinical cases. Clinical samples were examined histologically to determine whether they were benign or malignant and, if malignant, whether they had evidence of local invasion or regional metastasis. Canine fibroblast cultures and normal canine mammary epithelial tissues were used as reference standards for cell lines and mammary tumors, respectively. SAMPLE POPULATIONS: 28 canine mammary tumor tissue samples obtained surgically from clinical cases and samples from 7 canine mammary tumor cell lines derived from primary canine mammary tumors. PROCEDURE: c-erbB-2 mRNA levels were determined by means of hybridization of total polysomal RNA with a 32P-labeled human c-erbB-2 probe on dot blots, and results were quantified by means of scanning densitometry. Overexpression of c-erbB-2 was defined as an autoradiographic density > or = 2 times the density of reference samples on the same blot. RESULTS: Overexpression of c-erbB-2 was detected in 17 of 23 malignant tumors, 0 of 5 benign tumors, and 2 of 7 mammary tumor cell lines. c-erbB-2 overexpression was correlated with a histopathologic diagnosis of malignancy (P = 0.005) but not with the presence of local invasion or regional metastatic disease (P = 0.621). CONCLUSIONS: Results suggest that overexpression of c-erbB-2 occurs prior to the development of metastatic disease in canine mammary tumors and plays a role in the development of malignancy.

Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence of hG1.16 and the published rat L37 sequence. Southern-blot analysis revealed that hG1.16 exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.

Selective induction of cell cycle regulatory genes cdk1 (p34cdc2), cyclins A/B, and the tumor suppressor gene Rb in transformed cells by okadaic acid.

Genes encoding cdk1 (p34cdc2), cyclin A, cyclin B, and the tumor suppressor gene Rb are fundamental regulators of cell cycle progression which associate as a complex with the transcription factor E2F. Expression of many of these proteins has previously been shown to be repressed by okadaic acid, a specific inhibitor of protein phosphatases 1/2A (PP1/PP2A), resulting in growth arrest in nontransformed but immortalized cells. We have investigated levels of mRNA encoding cdk1 (p34cdc2), cyclin A, cyclin B, Rb, GAPDH, c-myc, and histone H4 genes for sensitivity to okadaic acid in HeLa cells to determine if transformation altered their regulation. Serum starvation slowed growth and diminished mRNA levels for all genes tested except c-myc and GAPDH. When starved cells were subsequently exposed to 19 nM okadaic acid or refed 10% serum, mRNA levels of cyclin A, cyclin B, cdk1, and Rb dramatically increased while mRNA levels for c-myc and GAPDH were largely unaffected. Histone H4 mRNA levels and the rate of DNA synthesis were greatly enhanced by serum addition but not affected appreciably by okadaic acid. Okadaic acid was also effective in blocking proliferation of exponentially growing HeLa cells at G2/M and S phase. Despite the cell cycle phase-specific block, elevated mRNA levels for cdk1, cyclin A, cyclin B, Rb, and suppression of H4 mRNA levels were detected and persisted for at least 12 hr following okadaic acid removal. The results demonstrate that cell cycle progression is blocked and several cell cycle regulatory genes, encoding transcription factor E2F-associated proteins, experience elevation of mRNA levels through mechanisms sensitive to okadaic acid likely through a PP1/PP2A-sensitive mechanism. Data from transformed cells contrast with data from immortalized but nontransformed cells in which okadaic acid also blocks cell cycle progression during G2/M phase but suppresses expression of these genes. Such contrasts may be correlated with reduced growth factor dependence and transformation.

Immediate-early transcription from the channel catfish virus genome: characterization of two immediate-early transcripts.

With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level approximately six times that of ie2 in cycloheximide-treated cells. The putative product of ORF 9 is predicted to have a basic pI and contains a potential zinc-binding domain, making it a probable transcription factor. ORF 8a encodes a putative product which is very hydrophobic, an unusual characteristic for an IE protein.

Overexpression of c-fos induces expression of the retinoblastoma tumor suppressor gene Rb in transfected cells.

Hypophosphorylated Rb, the product of the tumor suppressor gene associated with hereditary retinoblastoma, is thought to act as a suppressor of cell growth and proliferation during G1 phase. We investigated whether Rb expression was dependent on the expression level of the immediate early cell growth gene, c-fos, which is transiently expressed as cells re-enter G1 phase from quiescence. To explore the functional relationship between c-fos and Rb, a eukaryotic expression plasmid was constructed containing the c-fos gene under control of the SV40 promoter complex. This plasmid was co-transfected with plasmids encoding pRSV cat and G418 resistance, into HeLa S3 cells, and clonal populations of transfected (RSfos) cells selected. High levels of c-fos expression in transfected cells were confirmed by both western and northern blot. Rb protein content per cell was determined by flow cytometry using an Rb-specific primary antibody and a FITC-conjugated secondary antibody. Higher expression of Rb (2-6 fold/cell) in approximately 20% of RSfos transfected cells was observed in comparison to parental HeLa cells. Rb content per cell increased approximately 2-fold during the cell cycle in both parental HeLa cells and RSfos cell clones which overexpressed Rb. Rb accumulation occurred in a manner consistent with normal mass accumulation during the cell cycle. Overexpression of Rb in RSfos cells was also confirmed by western blot analysis. Because one possible function of RB may be to act as a brake on cell growth, it is possible that overexpression of RB acts as an inhibitory counter activity to overexpression of the growth promoting activity of c-fos. This possible balancing activity of Rb was further suggested when Rb protein expression levels were measured in different clonal lines of RSfos transfected cells overexpressing increasing levels of c-fos. Overexpression of Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb expression may be regulated in a manner which balances the transcription stimulatory effects of c-fos overexpression and its effects on the transcription of other genes during the cell cycle.

Neurotransmitter receptor agonists regulate growth hormone gene expression in cultured ovine pituitary cells.

The regulation of growth hormone (GH) secretion and GH mRNA content by the dopaminergic agonist, bromocriptine (BRO); the beta-adrenergic agonist; isoproterenol (ISO); the alpha 1-adrenergic agonist, methoxamine (MET); the alpha 2-adrenergic agonist, clonidine (CLON); the serotonergic agonist, quipazine (QUIP); somatostatin (SS) and GH-releasing hormone (GHRH) were studied using cultured ovine anterior pituitary cells. Clonidine and BRO (10(-6) M) inhibited basal and GHRH (10(-10) M)-stimulated GH release. Bromocriptine enhanced GH mRNA content and potentiated the GHRH (10(-8) M)-stimulated content of GH mRNA, while CLON had no effect on GH mRNA. Quipazine had little effect on GH secretion and no effect on GH mRNA content. Methoxamine and ISO (10(-6) M) increased basal secretion of GH and both enhanced GHRH-stimulated GH secretion. Both MET and ISO increased GH mRNA content of cultured ovine pituitary cells. Somatostatin (10(-7) M) inhibited GHRH-stimulated GH secretion and GH mRNA accumulation. These results support the hypothesis that neurotransmitters may regulate or interact to further modulate pituitary hormone release. Moreover, the data indicate that neurotransmitters may not only regulate secretion but also regulate GH mRNA content and thus affect hormone synthesis.

c-fos expression is required during all phases of the cell cycle during exponential cell proliferation.

Transient transcription of the c-fos gene is induced by serum stimulation of quiescent cells during the earliest part of Gl phase, reaching maximum levels of mRNA within 30 min. To determine whether expression of c-fos is required, or has any regulatory role in continuous exponential cell proliferation following re-entry into the cell cycle, a chimeric plasmid was constructed containing the human c-fos gene such that transcription was under control of the SV40 promoter complex. The plasmid was co-transfected into HeLa S3 cells (RSfos cells) along with plasmids encoding pRSVcat and G418 resistance followed by clonal propagation. The regulatory role of c-fos in an exponentially growing transfected RSfos cell clone (CP17-14) and parental HeLa cells was assessed through studies of c-fos overexpression or suppression of c-fos translation by treatment with a c-fos antisense 16-mer oligonucleotide. Transfected cells grew normally despite excess expression of c-fos. In contrast, antisense oligonucleotide treatment efficiently suppressed proliferation in normal exponentially growing cells by more than 70% for approximately 60 hr. Beyond this time oligonucleotides were ineffective likely due to degradation/depletion. In contrast, transfected cells grew normally, indicating overexpression of c-fos was sufficient to neutralize the effects of c-fos antisense oligonucleotides. Flow cytometric analysis of cell cycle phase distribution and determination of proliferation rate in oligonucleotide treated HeLa cells revealed a virtually complete inhibition of cell proliferation without a block in any specific cell cycle phase. In addition, no effect of oligonucleotide treatment on cell cycle phase distribution was observed in CP17-14 cells. Overexpression of c-fos rendered these cells resistant as the antisense c-fos oligonucleotides were unable to impose proliferative inhibition. These results demonstrate that c-fos expression is required during all phases of the continuous cell cycle in exponentially growing cells suggesting an important maintenance role for c-fos in addition to its role during re-entry into the cell cycle from quiescence.

Optimization of subtractive hybridization in construction of subtractive cDNA libraries.

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C(o)t values.

Cloning and partial sequence analysis of a Mycoplasma synoviae DNA fragment encoding epitopes shared with the major adhesin P1 protein of Mycoplasma pneumoniae.

Polyclonal antibodies specific for the adhesin P1 protein of Mycoplasma pneumoniae were used to screen an expression library of M. synoviae genomic DNA constructed in the expression vector lambda gt11. Following several cycles of immunoscreening using the anti-P1 antiserum, a lambda gt11 recombinant clone containing 3.9 kilobase pairs (kbp) of M. synoviae DNA was identified and isolated from the expression library. Expression of the recombinant clone (designated MS-1) in Escherichia coli Y1089 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude E. coli lysates revealed the presence of two novel proteins. Two antibodies that recognize the adhesin polypeptide--chicken anti-M. synoviae antibodies and anti-P1 antiserum--reacted with both proteins on immunoblots. Partial sequence analysis of the M. synoviae DNA in clone MS-1 and computer comparison of the predicted amino-acid sequences with existing protein sequence files revealed homology with the adhesin P1 protein of M. pneumoniae.

Overexpression of c-erbB-2 and c-myc but not c-ras, in canine melanoma cell lines, is associated with metastatic potential in nude mice.

Overexpression of the proto-oncogenes c-erbB-2, c-myc, and c-ras have been associated with neoplastic transformation in a variety of tumours. We investigated expression of these oncogenes in 5 canine melanoma cell lines and 6 clonal derivatives of 1 of the cell lines, CML-6M, to determine what impact overexpression had on tumour cell growth and metastatic potential. All 11 cell lines were tumourigenic at subcutaneous inoculation sites in nude mice, but spontaneous metastasis to lung was a characteristic of only the CML-6M cell line and 3 of 6 clonal derivatives of CML-6M. Investigation of oncogene overexpression revealed no obvious pattern of expression among the 5 tumour-derived cell lines whereas overexpression of c-erbB-2 and c-myc was consistently found in the 3 clonal cell lines characterized by high metastatic potential, and in primary and metastatic mouse xenografts induced by these lines. This data suggests involvement of overexpression of these genes in development of canine melanoma and associates their overexpression with metastatic potential in nude mice.

Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library.

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71-95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.

Development of a DNA probe for identification of bovine herpesvirus 4.

A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sensitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 micrograms of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.

Growth of HeLa S3 cells cotransfected with plasmids containing a c-fos gene under the control of the SV40 promoter complex, pRSVcat, and G418 resistance.

HeLa S3 cells, which have been fractionated into sequential and synchronous cell cycle phase-specific fractions, express c-fos at twice the basal levels in the earliest part of G1 phase. To determine whether this peak in c-fos synthesis has regulatory significance, a DNA construct was prepared which contained the human c-fos gene under the transcriptional control of the SV40 promoter complex. The pc-fos(human)-1 gene (9 kilobases) was inserted into the eukaryotic expression vector pSG5 (4.076 kilobases) at the EcoRI site. Electroporation with an exponentially decaying pulse was employed to cotransfect this construct into HeLa S3 cells along with the plasmids pRSVcat and the neomycin-resistance plasmid pF beta fos3' neo. The level of transient expression of each plasmid was determined. Transfection efficiency was determined as percentage fluorescent cells by measurement of immunofluorescence with a chloramphenicol acetyltransferase (CAT) antibody. Efficiency of transfection ranged up to approximately 5% of the cells. Transfected cells were selected on the basis of resistance to Geneticin (G418) at 400 micrograms/mL. CAT fluorescence and Geneticin resistance were employed to select permanently transformed cell lines. Compared with exponentially growing cells, successfully transfected cell lines expressed more than twice the level of c-fos mRNA as determined by dot-blot analysis and 16 times more of the 62-kilodalton c-fos protein as determined by Western blot analysis. As all cells in the population were not stable c-fos transfectants, this value is likely to be an underestimate of the overexpression level. In addition, expression was under the control of a strong serum induction insensitive promoter, unlike the native c-fos promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Quick and high-efficiency electroelution of nucleic acid fragments.

We describe a quick, simple, and inexpensive technique for the electroelution of nucleic acid fragments that provides a high yield of DNA by using common laboratory components. The quantity of buffer used for the recovery of nucleic acid fragments is relatively small, the quality of the DNA recovered is relatively high, and more than one electroelution can be carried out at the same time.

Expression of growth hormone, growth hormone releasing hormone, and somatostatin genes is unperturbed in the streptozotocin-induced diabetic rat.

The effects of streptozotocin diabetes on the level of growth hormone, growth hormone releasing hormone, and somatostatin mRNA was measured in control rats, in diabetic rats maintained on insulin, and in diabetic rats in which insulin had been withheld for 3 days. Total cytoplasmic RNA samples were prepared from the pituitary and hypothalamic tissues of each animal and analyzed by dot blot or Northern blot hybridization. No significant difference was observed between control and insulin-treated groups with regard to body weight or plasma glucose concentration. The insulin withdrawal group had significantly higher plasma glucose concentrations and lower body weights, confirming diabetic status. There was no significant difference in the level of growth hormone, growth hormone releasing hormone, and somatostatin mRNA among any of the three groups however. We conclude that alterations in the regulation of circulating growth hormone in the streptozotocin-induced diabetic rat, removed from insulin treatment for 3 days, did not occur at the transcriptional or RNA processing level. This conclusion extends to hypothalamic growth hormone releasing hormone, and somatostatin gene expression as well. Regulatory changes in growth hormone level previously noted during insulin withdrawal in the streptozotocin-induced diabetic rat could be the result of post-transcriptional processes operating at the level of hormone synthesis or release.