Separation Of Polypeptides Research Articles

Immunogenic Proteins Characterization in the Larvae of Oestrus ovis by Western Blot Analysis

The present study was undertaken to characterize and identify immunogenic proteins of Oestrus ovis in Telangana. Oestrus ovis is the most predominant nasal bot fly causing nasal myiasis in India. The collected larvae from Jiyaguda slaughter house, Hyderabad are seperated into L2 and L3 larvae were washed with normal saline and PBS pH 7.4 and preserved at -70°C for antigen preparation and some were preserved in 70 % ethanol for identification. The larvae were speciated by light microscopic examination based on morphological features such as ventral spines, anterior hooks and “D” shaped posterior spiracles. In this study, an antigen was prepared from second and third instar larvae of Oestrus ovis using a soluble buffer solution. The resulting Crude Somatic Larval (CSL) antigen had a protein concentration of 27.4 mg per ml. The CSL antigen was resolved in 8% SDS-PAGE, Immunoblotting was performed by electro transferring the separated polypeptides onto a nitrocellulose (NC) membrane. Treatment with specific reagents and antibodies resulted in the identification of 19 immunoreactive polypeptides, with molecular weights matching those observed in SDS-PAGE. Four polypeptides with molecular weights of 58.56, 20.74, 19.09, and 28.91 kDa exhibited the strongest reaction towards the antibodies raised against CSL antigen. These four polypeptides are considered promising candidates for future immunizations and testing their protective efficacy through in vivo assays. These findings have implications for advancing strategies to combat myiasis, a parasitic disease caused by Oestrus ovis larvae.

Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein.

Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

Humoral immune response of Galleria mellonella after repeated infection with Bacillus thuringiensis.

The insect immune system relies on innate mechanisms only. However, there is an increasing number of data reporting that previous immune challenge with microbial elicitors or a low number of microorganisms can modulate susceptibility after subsequent lethal infection with the same or different pathogen. This phenomenon is called immune priming. Its biochemical and molecular mechanisms remain unravelled. Here we present that Galleria mellonella larvae that survived infection induced by intrahemocelic injection of a low dose of Bacillus thuringiensis were more resistant to re-injection of a lethal dose of the same bacteria but not other bacteria and fungi tested. This correlated with enhanced activity detected in full hemolymph as well as in separated hemolymph polypeptides. In addition, we observed differences in the hemolymph protein pattern between primed and non-primed larvae after infection with the lethal dose of B. thuringiensis. Expression of genes encoding inducible defence molecules was not enhanced in the primed larvae after the infection with the lethal dose of B. thuringiensis. It is likely that priming affects the turnover of immune related hemolymph proteins; hence, upon repeated contact, the immune response may be more ergonomic.

Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS.

A laboratory exercise illustrating the sensitivity and specificity of Western blot analysis

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the separated polypeptides from the gel is created on the membrane, which is then probed with antibodies or other ligands to identify specific polypeptide(s). Here, we report an undergraduate laboratory exercise involving Western blotting. During the 3-week laboratory exercise, students investigated the likelihood of the presence of a serum albumin in fruit fly (Drosophila melanogaster) homogenate, bovine calf serum (BCS), and fetal bovine serum (FBS). Students isolate proteins from fruit fly larvae and perform sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic blotting, and immunoassay comparing those proteins with BCS and FBS proteins. Their results indicate that serum albumin is present in BCS and FBS but is absent in fruit flies. In the process, the specificity and sensitivity of Western blot analysis is demonstrated. The laboratory exercise can be easily incorporated into any college-level biochemistry or molecular techniques laboratory. The procedure used can be easily adapted to study other proteins.

Comparative Immunogenicity of Human Immunodeficiency Virus Particles and Corresponding Polypeptides in a DNA Vaccine

The immunogenicity of a plasmid DNA expression vector encoding both Gag and envelope (Env), which produced human immunodeficiency virus (HIV) type 1 virus-like particles (VLP), was compared to vectors expressing Gag and Env individually, which presented the same gene products as polypeptides. Vaccination with plasmids that generated VLP showed cellular immunity comparable to that of Gag and cell-mediated or humoral responses similar to those of Env as immunization with separate vectors. These data suggest that DNA vaccines encoding separated HIV polypeptides generate immune responses similar to those generated by viral particles.

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.

Long-chain class III metallothioneins as a mechanism of cadmium tolerance in the marine diatom Phaeodactylum tricornutum Bohlin

Cadmium tolerance of the marine diatom Phaeodactylum tricornutum Bohlin was studied. P. tricornutum growth was significantly reduced by cadmium concentrations of 5 mg l −1 or higher. The EC 50 value was 22.39 mg l −1 of cadmium after four days of exposure. Therefore, P. tricornutum is more tolerant to cadmium than other microalgal cells. In response to exposure to cadmium, P. tricornutum synthesize class III metallothioneins which sequester the metal in a harmless form. However, cadmium tolerance of P. tricornutum cells is not only due to the ability of this microalga to synthesize class III metallothioneins (γ-Glu-Cys) n -Gly, but also because these metallothioneins are composed of long-chain polypeptides. The cadmium-metallothionein complexes of P. tricornutum cells contain predominantly polypeptides of n = 4−9. Long-chain metallothioneins are more efficient in binding heavy metals. Capillary electrophoresis was used to separate class III metallothioneins. This is a recent technique used to separate many molecules. With this technique, the length of the separated polypeptides can also be estimated.

Renal cell carcinoma and normal kidney protein expression.

Renal cell carcinoma (RCC), a human kidney cancer from the proximal tubular epithelium, accounts for about 3% of adult malignancies. Molecular and cytogenetic analysis have highlighted deletions, translocations, or loss of heterozygosity in the 3p21-p26, a putative RCC locus, as well as in 6q, 8p, 9pq, and 14pq. Studies on phenotypic expression of human kidney tissue and on post-translational modifications in RCC have not yet provided a marker for early renal cell carcinoma diagnosis. Current diagnostic methods do not help to detect the tumor before advanced stages. We therefore used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to study normal and tumor kidney tissues in ten patients suffering from RCC. A human kidney protein map in the SWISS-2DPAGE database accessible through the ExPASy WWW Molecular Biology Server was established. Of 2789 separated polypeptides, 43 were identified by gel comparison, amino acid analysis, N-terminal sequencing, and/or immunodetection. The comparison between normal and tumor kidney tissues showed four polypeptides to be absent in RCC. One of them was identified as ubiquinol cytochrome c reductase (UQCR), whose locus has elsewhere been tentatively assigned to chromosome 19p12 or chromosome 22. A second polypeptide was identified as mitochondrial NADH-ubiquinone oxido-reductase complex I whose locus is located on chromosome 18p11.2 and chromosome 19q13.3. These result suggest that the lack of UQCR and of mitochondrial NADH-ubiquinone oxidoreductase complex I expression in RCC may be caused by unknown deletions, or by changes in gene transcription or translation. It might indicate that mitochondrial disfunction plays a major role in RCC genesis or evolution.

The 160k alpha 1(IV) chain, a short form of a type IV collagen polypeptide, of bovine lens capsule retains the NC1 domain.

We recently reported that the bovine lens capsule contained a shorter alpha 1(IV) chain (160k) as a major polypeptide in addition to the 180k alpha 1(IV) chain [J. Biochem. (1995) 117, 1298-1304]. Two experiments were performed to examine whether or not the 160k polypeptide retained the carboxyl-terminal NC1 domain. On immunoblotting analysis with a monoclonal antibody (H11) raised against the NC1 domain of the human alpha 1(IV) chain [positions 1643-1650; near the carboxyl-terminal end of the human alpha 1(IV) chain], the 180k and 160k polypeptides showed identical immunoreactivity, suggesting that the two chains had the same human alpha 1(IV) collagen NC1 domain sequence. Another monoclonal antibody (H21) specific for the NC1 domain of human alpha 2(IV) did not react with these polypeptides, but with the bands corresponding to 175k and 155k. The 160k polypeptide was selectively solubilized from bovine lens capsules, leaving the other major polypeptides, 180k and 175k, insoluble. The 160k polypeptide was separated by preparative electrophoresis. Bacterial collagenase digestion of the separated 160k polypeptide produced collagenase-resistant segments of about 29k and 30k in size based on globular standards. These sizes corresponded well with those of the NC1 domains of type IV collagen alpha chains (25-30k). The results indicated that the 160k polypeptide retained the carboxyl-terminal NC1 domain of the alpha 1(IV) chain. In turn, the 20k polypeptide of the amino-terminal region or the 7S domain of 180k alpha 1(IV) would have been excised to yield 160k alpha 1(IV), assuming that the 160k alpha 1(IV) chain is a processed form of the 180k alpha 1(IV) one and not an alternatively spliced chain of the alpha 1(IV) gene.

Schistosoma mansoni: Excretory-Secretory Polypeptides Exhibit Selective Binding to Plasma Components of the Snail Biomphalaria glabrata

Previous studies have shown that Schistosoma mansoni excretory-secretory polypeptides (ESP) inhibit various internal defense functions of hemocytes from Biomphalaria glabrata and that plasma also may exert a modulatory effect on hemocyte activity, To better understand how plasma may influence hemocyte-schistosome interactions in inbred strains of snails, biotinylated ESP (b-ESP) were used as probes to identify ESP-reactive plasma components and partially characterize the nature of their binding interactions. In a plasma binding assay, b-ESP bound in a dose-dependent fashion to immobilized snail plasma, although no quantitative differences in ESP reactivity to plasma of schistosome-susceptible (M-line) and resistant (13-16-R1) snail strains were detected. Moreover, co-incubation of b-ESP with homologous plasma or pretreatment of plasma with nonbiotinylated ESP in the plate assay significantly reduced b-ESP binding to immobilized plasma, indicating a specific interaction between ESP and plasma. Pretreatment of b-ESP with mannose, porcine stomach mucin, fetuin, and asialofetuin resulted in a significant inhibition of b-ESP binding to plasma, whereas pretreatment of immobilized plasma with a cocktail of monosaccharides (including mannose), porcine stomach mucin, and fetuin had no inhibitory effect. These data suggest the presence of a carbohydrate-binding protein(s) in sporocyst ESP that is targeted to plasma glycoconjugates. Based on the carbohydrate content of the major inhibitory glycoproteins, galactosyl and n-acetyl-galactosaminyl sugars may represent putative determinants for the lectin-like ESP molecule(s). However, ESP binding to plasma from M-line and 13-16-R1 B. glabrata strains exhibited similar sugar and glycoprotein inhibition patterns. SDS-PAGE and electroblot analyses further demonstrated that ESP bound to a subset of separated plasma polypeptides, most prominently to a doublet of 52 and 54 kDa, and a complex of proteins with molecular masses greater than 150 kDa. Other polypeptides exhibiting weaker binding interactions included components of 34, 60, 64, 86, and 125 kDa. Although both M line and 13-16-R1 snail strains contained a similar complement of ESP-binding plasma proteins, ESP binding to the 34- and 86-kDa proteins occurred at higher frequencies in susceptible M-line plasma. It is concluded that ESP, released during in vitro transformation of S. mansoni miracidia, contain carbohydrate-reactive proteins capable of selectively binding to components of snail plasma. Quantitative differences between snail strains in the occurrence of ESP-binding plasma molecules was documented.

Nuclear DNA Helicase II Unwinds both DNA and RNA

Nuclear DNA helicase II (NDH II) has been purified to near-homogeneity by exploiting its high affinity to poly[(rI).(rC)]-agarose. The purified enzyme was obtained as two catalytically active forms of 130- and 100-kDa molecular mass, respectively. After treatment with cyanogen bromide, the separated polypeptides displayed very similar digestion patterns. Thus, the 100-kDa form most likely is a proteolytic product of the 130-kDa polypeptide. For DNA unwinding, NDH II could use any of the four rNTPs or dNTPs with Km values between 20 and 100 microM. DNA unwinding was stimulated up to 20-fold by substrates that contained single-stranded 3'-tails. NDH II-catalyzed DNA unwinding was strongly inhibited by RNA, but was little affected by DNA. The strongest RNA inhibitor, poly[(rI).(rC)], was also the strongest effector of the NTPase activity of NDH II. The binding constant for poly[(rI).(rC)] binding was about 2 x 10(7) M-1; the minimal binding site size was determined as 16 nucleotides. In agreement with its high affinity to RNA, NDH II unwound double-stranded RNA. RNA unwinding required the presence of a nucleoside triphosphate and a divalent cation (Mg2+). Thus, like the prototypic replicative helicase large T antigen of simian virus 40, NDH II may function in both DNA and RNA unwinding.

Secondary structural changes of N-bromosuccinimide-cleaved bovine serum albumin in solutions of sodium dodecyl sulfate, urea, and guanidine hydrochloride

Two tryptophyl peptide bonds of bovine serum albumin (BSA), Trp 134-Gly 135 and Trp 212-Ser 213, were cleaved with N-bromosuccinimide (cleaved BSA). The relative extents of secondary structures in cleaved BSA were determined by curve-fitting of the circular dichroism spectrum. Cleavage at these two sites caused an appreciable disruption in the helicity of the protein. The cleaved BSA contained 55% α-helical structure as against 66% for the intact BSA. The cleavage also made the helical structures unstable in solutions of urea and guanidine hydrochloride. The extent of α-helical structure of cleaved BSA decreased to 50% in sodium dodecyl sulfate (SDS) solutions. However, some moieties, the helical structures of which had been disrupted by N-bromosuccinimide cleavage, appeared to re-form the helical structure in SDS, although the cleavage effect remained through the urea and guanidine denaturations and also through the thermal denaturation. On the other hand, the three resultant polypeptides, Asp 1-Trp 134, Gly 135-Trp 212, and Ser 213-Ala 582, were separated by reduction of the disulfide bridges joining them. These three separated polypeptide fragments showed only 20% helix content. However, the extent of the helical structure in the three polypeptide fragments increased to 35–40% in SDS. Secondary structural changes in the three fragments were also examined in urea and guanidine hydrochloride solutions.

Immunization against hepatitis B

Hepatitis B surface antigen in the form of 22-nm spherical particles (and tubular forms) is excess virus coat protein. Guidelines for the preparation of the 22-nm spherical particles (and their separated polypeptides) derived from the plasma of asymptomatic human carriers were suggested by the WHO Expert Committee on Viral Hepatitis in 1977, and the proposed requirements for the 22-nm hepatitis B particle vaccine were published by the WHO Expert Committee on Biological Standardization in 1981 and revised in 1983. Such preparations have been tested for safety and protective efficacy, and many clinical studies in several million individuals with the plasma-derived vaccine have demonstrated the immunogenicity, high protective efficacy and safety of the currently licensed preparations meeting WHO requirements (Deinhardt and Zuckerman 1985).

A possible ligand of serum origin for the kidney autoantigen of Heymann nephritis.

Earlier studies have localized the Heymann nephritis (HN) autoantigen (gp330) in the coated pits of the plasma membrane and multivesicular bodies of the glomerular epithelial cell. Because of these locations in the glomerular epithelial cells, it has been suggested that the HN Ag may be a receptor. The aim of our study was to search for a ligand which can bind the HN autoantigen. Normal rat serum was subjected to SDS-PAGE under reducing and non-reducing conditions followed by Western analysis of the separated polypeptides. A reaction was revealed directly by autoradiography using 125I labeled HN autoantigen as a probe and indirectly by enzyme immunodetection using unlabeled nephritogenic autoantibody (anti-gp330) eluted from glomeruli of diseased rats followed by biotinylated rabbit anti-rat IgG avidin-peroxidase complex. A polypeptide of 76 kDa Mr was identified under non-reducing conditions as a serum protein reacting with the HN autoantigen. Reactivity of the 76-kDa polypeptide was lost when serum was electrophoresed under reducing conditions. Direct binding of the 76-kDa polypeptide obtained from serum to the HN autoantigen obtained from kidney suggests that the 76-kDa polypeptide may be a ligand for the autoantigen. This is the first documentation of a possible ligand for the HN autoantigen. Not only does this polypeptide bind to the HN autoantigen but it also shows direct binding with the nephritogenic autoantibody eluted from glomerular deposits. This characteristic of the 76-kDa polypeptide indicates that this serum protein may potentially play a role in the development of the glomerular lesion of active HN. Further analysis of this serum component should assist in understanding the normal function of the HN autoantigen.

Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis

A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and—because there is no protein fixation step—nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Schägger and G. Von Jagow (1987), Anal. Biochem., 166, 368–379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.

Molecular analysis of alcohol dehydrogenase electromorphs in wild type and transformed [formula omitted

The protein expressed by the alcohol dehydrogenase locus ( Adh ) in D melanogaster comprises a small group of electromorphs. We are able to study the expression of these electromorphs by electrophoretic separation and subsequent probing of blots of the separated polypeptides with antiserum for alcohol dehydrogenase (ADH). In the present study we have utilized this technique to study and compare the ADH electromorphs in wild type D. melanogaster with D. melanogaster transformants which carry an Adh gene from D. grimshawi , D. hawaiiensis or D. affinidisjuncta and produce functional ADH (10,19). We have determined that polypeptides are produced by the donor loci in the transformed flies and further show that although the molecular weight of the expressed polypeptides is similar to D. melanogaster electromorphs, the isoelectric points are not similar. Thus this methodology offers the potential to study naturally occuring ADH electromorphs and null alleles independent of enzymatic activity assays.

Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.

Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.

Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.

Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.

DNA polymerase alpha associated primase from rat liver: physiological variations.

A primase activity associated to DNA polymerase alpha from rat liver is described. Both activities were absent in normal adult rat liver but were concomitantly induced after partial hepatectomy. As previously shown for polymerase alpha and DNA topoisomerase II, primase activity reached a maximum value 40-43 h after the partial removal of the liver. Primase activity was shown to catalyze dNMP incorporation on unprimed single-stranded DNA template (M13 DNA) in the presence of rNTP. The activity was not detectable on poly(dA) or poly(dG) but was efficient on poly(dT) or poly(dC). However, the reliability of the primase assay in the presence of poly(dC) was dependent upon the degree of purification of the enzyme. The ribo primers were about 10 nucleotides long, and the reaction was completely independent of alpha-amanitin, a strong inhibitor of RNA polymerases II and III. Primase and polymerase were found tightly associated. A cosedimentation on a 5-20% sucrose gradient was always obtained, independent of the ionic strength. There was also a close coincidence between alpha-polymerase and primase activities during phosphocellulose, hydroxylapatite, and single-stranded DNA Ultrogel chromatography. It has been previously demonstrated by us and others that primase and alpha-polymerase are on separated polypeptides. The association of two activities in the replication complex and the conditions allowing their separation are discussed.