Polylinker Region Research Articles

Expression of LDL receptor, apolipoprotein B, apolipoprotein A-I and apolipoprotein A-IV mRNA in various mouse organs as determined by a novel RNA-excess solution hybridization assay

We report expression of LDL receptor, apolipoprotein B (apoB), apolipoprotein A-I (apoA-I) and apolipoprotein A-IV (apoAIV) mRNA in various mouse organs. These mRNA were quantified by an RNA-excess solution hybridization assay. For preparing specific probes, we cloned cDNA fragments of rat LDL receptor, apoB and apoA-I and mouse apoA-IV into the polylinker region of pGEM3Zf(+) and used the recombinant vectors for preparing 32P-labeled cRNA probes as well as RNA standards using the T7 and SP6 promoters flanking the polylinker regions. Preparation of cRNA probes and RNA standards is faster and more convenient than preparing cDNA probes and ssDNA standards. Absolute levels of mRNA were quantified in the liver, intestine, kidney, heart, lung, spleen and adrenals of females of two mouse strains. C3H/HeJ and C57BL/6J. ApoB, apoA-I and apoA-IV genes in mice are expressed in the liver and intestine and LDL receptor gene is expressed mainly in liver, intestine and adrenals. ApoA-I mRNA levels were found to be 730 and 1039 molecules per cell in liver and intestine, respectively, in C3H mice and 762 and 952 molecules per cell in C57BL mice. ApoB mRNA levels were 66 and 170 molecules per cell in the liver and intestine of C3H and 83 and 243 molecules per cell in C57BL, respectively. ApoA-IV mRNA was found to be 3525 and 2964 molecules per cell in the liver and intestine of female C57BL mice, respectively. LDL receptor mRNA levels were 39, 32 and 14 molecules per cell in the liver, intestine and adrenals of C3H.

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli

Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5′ end of the genes, and two ochre stop codons have been created at their 3′ ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector eaturing a tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.

What is a genus in the Trypanosomatidae family? Phylogenetic analysis of two small rRNA sequences.

The family Trypanosomatidae of the order Kinetoplastida is characterized by flagellated protozoa that present a kinetoplast. Most of the recognized genera are parasitic protozoa and, depending on host range, have been classified as either monogenetic trypanosomatids (parasitic in a single invertebrate) or digenetic trypanosomatids (parasitic in both invertebrate and vertebrate hosts). Trypanosomatids are unusual eukaryotes in that their nuclear large-subunit t-RNA genes reveal a complex organization with the occurrence of internal transcribed spacers separating the coding regions for two large (a and B) and five small (Sl-S4 and S6) rRNA molecules (White et al. 1986; Spencer et al. 1987; Hemindez et al. 1988 ) . The small rRNA molecule S3 corresponds to the characteristic eukaryotic 5.8s rRNA (Hasan et al. 1984). We have previously published the molecular cloning of rRNA genes from Trypanosoma cruzi (Hemandez et al. 1988) and determined its small-subunit (SSU) rRNA coding region ( Hemlndez et al. 1990). We report here the nucleotide sequences of T. cruzi S3 (or 5.8s) and S 1 rRNA coding regions and compare them evolutionarily with their published homologues from the monogenetic species Crithidia fasciculata (Spencer et al. 1987) and the digenetic species T. brucei (Campbell et al. 1987). In addition, our previous SSU rRNA sequence (Hemandez et al. 1990) was further analyzed with the inclusion of six of nine variable regions and with Dictyostelium discoideum (Sogin et al. 1986) as a second outgroup. Appropriate restriction fragments from our genomic rDNA clone pRTC42 (Hernandez et al. 1988) were subcloned into the polylinker region of M 13mp18 and M13mp19 vectors (Yanisch-Perron et al. 1985). Nucleotide sequencing was carried out from single-stranded templates by the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Tabor and Richardson 1987) by using the SequenaseM kit (U.S. Biochemical Corp.) and (u-~~P) dATP ( Amersham). The 5’ and 3’ ends of Sl and S3 rRNA coding regions were assigned by comparison with published sequences from C. fasciculata (Spencer et al. 1987) and from T. brucei (Campbell et al. 1987) (fig. 1). Sl rRNA, whose coding region did not exhibit 5’ terminal identity with the homologous regions of C. fasciculata and T. brucei, was partially sequenced from 5 ‘kinased RNA molecules (‘Y-~~P) ATP (New England Nuclear), after the partial enzymatic digestion technique using the Bethesda Research Laboratories RNA sequencing kit and protocol. An analogous genome sequence has been recently published from a Brazilian T. cruzi strain (Vieira de Arruda et al. 1990). Our direct rRNA sequence places the S 1 rRNA 5 ’ terminal coding region two nucleotides downstream from its indirect comparative assignment. The rest of the sequence is identical to ours, with the exception of an extra G in a run of five G’s that we find in positions 128132. This addition could be a natural polymorphism among strains. The trypanosomatid S3 and S 1 rRNA molecules correspond, respectively, to the rRNA 5.8s and to the central region of domain IV of the large-subunit rRNA from other eukaryotes (Hasan et al. 1984; Lanversin and Jacq 1989). To root the phylo-

In situ hybridization for the study of gene expression in neuro‐otologic research

In situ hybridization histochemistry technology was developed for future application to neuro-otologic research. This method allowed the detection of cellular mRNA in tissue sections from the temporal bone or brainstem after cRNA/mRNA hybridization. To produce specific cRNA, single-stranded 35S-labeled cRNA (complimentary to target mRNA) is transcribed from commercially available plasmid vectors. These vectors contain promotor sequences for specific synthesis of RNA, and polylinker regions that will accept cloned DNA inserts for virtually any target nucleic acid sequence of interest. The protocol used in this research was optimized for studies that included concomitant immunohistochemical evaluation. The combination of in situ hybridization and immunohistochemistry provides the only method to correlate molecular information (gene expression) with biochemical or molecular markers, such as peptides or proteins (mRNA translation products) on individual cells in the temporal bone or brainstem. Using these techniques, we examined the distribution of the neuropeptide calcitonin gene-related peptide in rat temporal bone and brainstem sections using calcitonin gene-related peptide (CGRP) antisera and CGRP cRNA probes. We used in situ hybridization histochemistry with a cRNA probe complementary to the 3'-end noncoding sequence of the alpha CGRP mRNA and immunohistochemistry with a polyclonal antibody to the (TYR)CGRP23-37 to study the distribution of CGRP mRNA and CGRP-like immunoreactivity in the central and peripheral facial nerve. Numerous motoneuron cell bodies in the facial nucleus and accessory seventh nucleus and cell bodies in the gustatory geniculate ganglion were found to contain CGRP mRNA and the CGRP peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae

An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUC19. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.

Antigenic conservation of the 15,000-dalton outer membrane lipoprotein PCP of Haemophilus influenzae and biologic activity of anti-PCP antisera

A gene from Haemophilus influenzae encoding an outer membrane lipoprotein of about 15,000 daltons and which comigrates with the peptidoglycan-associated lipoprotein (PAL) of H. influenzae on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been previously reported and designated pcp gene, and its product has been designated PCP. in order to obtain specific immunologic probes for the analysis of PCP expression, cellular location, and antigenic conservation in H. influenzae, pcp was fused to the lac polylinker region of plasmid pUC19 and the hybrid gene was expressed in Escherichia coli. PCP purified from these cells was used to generate rabbit and mouse polyclonal antisera and mouse monoclonal antibody against PCP. Western immunoblot analysis with anti-PCP monoclonal antibody demonstrated that PCP is present and antigenically conserved in 30 tested strains of H. influenzae, including 27 clinical nontypeable strains. Polyclonal antiserum against PCP killed 9 of 11 clinical H. influenzae strains in a complement-mediated bactericidal assay, and bactericidal activity was additive with bactericidal activity of antisera against PAL. These results indicate that PCP is a potentially valuable component for a subunit vaccine against nontypeable H. influenzae disease, especially in combination with PAL or other components.

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence ▪ The novel enzyme in the presence of Mg 2+-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages φX174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a Polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.

High-efficiency oligonucleotide-directed plasmid mutagenesis.

A number of single- and double-base substitutions have been introduced into either the polylinker region or the lacZ gene in the plasmid vector pUC19. The efficiencies of these changes upon transfection of TG-1 bacterial cells were generally 70-80%. A strategy has been devised by which the wild-type DNA can be selectively destroyed. It is primarily based on the resistance of phosphorothioate internucleotide linkages to some restriction enzymes. A mismatch oligonucleotide is introduced into a gapped region and the gap is filled using three deoxynucleoside 5'-triphosphates and one deoxynucleoside 5'-[alpha-thio]triphosphate. Reaction with a restriction enzyme that is unable to hydrolyze phosphorothioates ensures that the DNA containing the mismatch oligonucleotide is only nicked. Concomitantly, the DNA that does not contain the desired mutation is linearized. Subsequent reactions with an exonuclease and DNA polymerase I yield mutant homoduplex DNA for transfection.

The repair patch ofE.coli(A)BC excinuclease

The size of repair patch made by E. coli DNA polymerase I (Poll) following the removal of a thymine-psoralen monoadduct by E. coli (A)BC excinuclease was determined by using an M13mp19 DNA with a single psoralen monoadduct at the polylinker region. Incubation of this substrate with (A)BC excinuclease, Poll and a combination of 3 dnTP plus 1 dNTP(alpha S) for each nucleotide, and DNA ligase resulted in a repair patch with phosphorothioate linkages. The preferential hydrolysis of phosphorothioate bonds by heating in iodoethanol revealed a patch size--with minimal nick translation--equal in length to the 12 nucleotide gap generated by this excision nuclease.

Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis

A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95–103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-β-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 μg/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.

Phagemid VPCS vectors for priming, cloning and sequencing

A phagemid was adapted for use as the vector in the vector-primer-cloner-sequencer cloning system. The use of this new vector markedly expanded the utility of this technology for the construction of cDNA libraries. Technological advantages and new capabilities include: (1) a greater number of unique restriction sites within the polylinker region; (2) the ability to produce single-stranded templates for nucleotide sequencing, and (3) a convenient means to synthesize strand-specific hybridization probes. With the use of this cloning system, a rat liver cDNA library (8.56 × 10 5 recombinants from 1 μg of poly(A) +RNA) was rapidly (in two days) constructed.

A Complete Protocol forIn SituHybridization of Messenger RNAs in Brain and Other Tissues With Radio-labeled Single-Stranded RNA Probes

We present a detailed protocol for In Situ hybridization of messenger RNA molecules in brain and other tissues, using 35S-labeled single-stranded RNA transcribed from commercially available plasmid vectors. These vectors contain promoter sequences for specific synthesis of RNA, and polylinker regions that will accept cDNA inserts for virtually any target molecule of interest. The protocol is optimized for studies that include concomitant irnmunohistochemical and cytoarchitectonic evaluations, and it is sufficiently detailed for the molecular biologist unfamiliar with histologic technique or the histologist inexperienced in molecular biological methods. We have used it with consistant results for In Situ hybridization studies using probes to more than twenty different mRNAs; and for several of those we have obtained results showing quantitative differences in experimentally altered mRNA levels. Included are details of perfusion fixation, tissue sectioning and preparation, probe synthesis, hybridization reaction, and signal detection. The introduction and annotated comments include rationale for basic procedures and the purpose of various specific steps in the hybridization histochemistry protocol. (The J HistotechnoL 12:169, 1989).

New bacteriophage vectors for the large scale production of single stranded insert DNA

SSEV 18 and SSEV 19, derivatives of the bacteriophages M13mp18/19, are new versatile cloning vectors allowing the large scale preparation of single stranded (ss) insert DNA. Replacing the original multiple cloning site by a synthetic 96 bp DNA fragment, a new polylinker region has been introduced containing complementary sequences designed to form a stem structure where single stranded insert fragments can be excised via a ‘master restriction’ site. The usefulness of such a vector has been demonstrated by the cloning of a 900 bp HindIII fragment derived from the Mycoplasma hyorphinis 23 S rRNA gene. After excision the single stranded insert was labeled isotopically and tested for sensitivity and specificity in detecting homologous sequences in pure DNA or cellular material immobilized on filters.

Development of a Monoclonal Antibody Specific for the Gamma Chain of the T-Cell Antigen Receptor Using an Open Reading Frame Expression Vector

To develop an anti-framework monoclonal antibody (mab) specific for the gamma (gamma)-chain of the T-cell antigen receptor (TCR), we expressed a part of the constant region of the gamma-chain (C gamma 2 gene segment) in E. coli using the pWR590 vector. This plasmid contains the E. coli lac promoter, operator, a truncated beta-galactosidase (beta-gal) gene (coding for the first 590 of the 1,007 amino acids of the beta-gal) and a polylinker region (at the 3' end of the beta-gal) containing nine restriction sites. These can be cleaved by any one of eight common restriction enzymes, permitting the introduction of the DNA fragment of interest. We employed the pT gamma 1 gamma-chain cDNA probe, which like the vast majority of the gamma-chain specific probes is aberrant and contains an in-frame stop codon at the junction of V and J regions. Computer analysis of the pT gamma 1 sequence revealed several MaeIII restriction sites that could result in a number of fragments. One of these fragments consisted of 245 base pairs (nucleotides 404-648) and contained most of the CI exon of the C gamma 2. Successful insertion of this fragment to the pWR590 vector was confirmed using restriction enzyme analysis. The C gamma insert was 12% of the construct. Expression of the pWR590-HpT gamma 1 recombinant plasmid in E. coli followed by SDS-PAGE analysis revealed a hybrid protein with a molecular weight of 85 kd which constituted at least 25% of the total E. coli insoluble protein. In contrast, cells transformed with the control pWR590 vector without insert expressed a 78 kd polypeptide chain. We developed several mabs against the pWR590-HpT gamma 1 hybrid protein by fusing spleen lymphocytes from BALB/c mice immunized with the pWR590-HpT gamma 1 protein, with cells of the NS1 mouse myeloma cell line. Screening of the mabs was carried out by ELISA against the pWR590-HpT gamma 1 hybrid protein and the control pWR590 beta-gal protein (beta-gal 590), derived by expressing in E. coli the pWR590 vector without gamma-chain insert. Two groups of mabs were obtained, those reacting with the pWR590-HpT gamma 1 hybrid protein only and those reacting with both the hybrid and the control beta-gal 590 proteins. The specificity of these mabs was further studied by Western blotting with similar results.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression vector pT7: TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase

A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene ( tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7: TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5 ' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from W thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7 : TKII vector provides a powerful approach with which: ( i) to investigate the translational and posttranslational regulation of the VV tk gene; ( ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and ( iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m 7G cap structure to the pT7 : TKII-derived VV tk RNA are presented.

A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase

We have developed a plasmid expression vector for the study of transcription terminators in Escherichia coli that utilizes the lux genes coding for the enzyme luciferase of the bioluminescent marine bacterium. Vibrio harveyi. The pBR322-derived plasmid, called pHV100, contains the E. coli lac promoter, the polylinker regions from the plasmid vector pUC18, and the V. harveyi lux genes. Insertion of transcription termination sites into the polylinker region results in decreased luciferase expression. Because the bioluminescence genes are not indigenous to E. coli, their expression can be studied in virtually any host strain without the complications of background activity. This facilitates sensitive measurements of terminator efficiency in hosts containing termination factor mutations. Bioluminescence can be easily monitored with high sensitivity, using a rapid photographic technique or a more quantitative photometric assay.

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

A series of nic − cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites ( AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUCIS and pUC19, while pMTL22 and -23 possess eleven new cloning sites ( AatII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNAI/RNAII region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic −, thus affording the lowest categorisation in genetic manipulation experiments.

Erythropoietin-β- D-galactosidase the generation, purification and use of a fusion protein

A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3′-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278. Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-β- D-galactosidase gene. After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-β- D-galactosidase were synthesized in the transformed bacteria. The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity. Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies. Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants. The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo. This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene.

Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia Coli

The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli 1M109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase a from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.