A microcosm study of the effect of Capitella sp. I (Polychaeta) population density on the mineralization of a pulse addition of the plasticizer DEHP (di[2-ethylhexyl]phthalate) demonstrated a 2-fold increase in mineralization (cumulative (CO2)-C-14 production) when worms were present. An additional experiment investigated the fate of ingested particle-bound C-14-DEHP and measured the loss of ingested C-14-DEHP into (CO2)-C-14, (DOC)-C-14 and (POC)-C-14 pools. Less than 1% of C-14 consumed during a 1 h ingestion period was ultimately respired as (CO2)-C-14, while 10% was excreted as (DOC)-C-14 and 89% as (POC)-C-14. Approximately 1% of ingested C-14 was retained in worm tissue 20 h after ingestion. Assuming density-independent feeding rates, worm respiration could account for 4.5% to 19.1% of the total microcosm (CO2)-C-14 production, suggesting that microbial respiration to (CO2)-C-14 was the dominant process. Pre-exposure of worms to DEHP (10 mu g g(-1) sediment dry wt) for 1 wk had no effect on the fate of ingested DEHP and distribution into the respective pools. Worms exerted a strong effect on ultimate DEHP degradation ((CO2)-C-14 production) but the effect was manifest at the lowest worm density and did not increase with increasing population size. The lack of an increased effect at greater population densities may be due to population density-dependent factors acting to decrease such important parameters as individual worm ventilation and feeding rates. A density-dependent decrease in feeding rate is supported by the observation that measured C-14 body burdens were highest at lowest worm densities.

We have isolated Bsp423I, a novel class-II restriction endonuclease from Bacillus species recognizing the palindromic sequence 5'-GCAGC-3' generating 5'-protruding tetranucleotides within the sequence complementary to 5'-GCAGC(N)g-3'. With respect to its isoschizomer Bbvl it can be isolated in higher purity and stability. A comparison of cleavage patterns obtained with Bsp423I using lambda, Ad-2, SV-40, phiX174, M13mpl9, pBR322, pBR328 and pUC18 DNAs of known nucleotide sequence (Figure 1, lanes 3-10) with computer-derived mapping data (1) predicts the sequence 5'-GCAGC-3'. The recognition sequence was confirmed by parallel digestion of lambda DNA with its isoschizomer Bbvl (2) (Figure 1, lane 2) resulting in both cases in fragments of approximately 1750, 1500, 1200, 1000, 970, 900, and numerous smaller bands which correlate with the computerderived length of 1741, 1538, 1523, 1249, 1246, 1242, 1027, 1020, 976, 974, 897, 894. 891 bp for the sequence 5'-GCAGC-3'. The exact positions of the cut 3' of the Bsp423I-recognition site were determined according to the enzymatic sequencing approach described in (3). An pBluescript Sk derivative with an insert containing a Bsp423I cleavage site was used for enzymatic sequencing reactions starting with a 5'-phosphorylated reverse M13 sequencing primer (CAGGAAACAGCTATGACC). In a parallel reaction, the same primer, [P]-endlabelled with T4 PNK and [Y3 P]ATP, was annealed to the template and the labelled primer was extended by treatment with Klenow enzyme and all four dNTPs through the Bsp423I site. The double stranded DNA was used as substrate for Bsp423I to produce an 5'-endlabelled DNA fragment comparable to the sequencing ladder. Samples were analyzed without or with ( / + ) further incubation with T4 DNA polymerase and all four dNTPs by electrophoresis and subsequent autoradiography (Fig. 2). In the Bsp423l reaction the observed single band comigrated with the 12th nucleotide 3' to the recognition sequence; after T4 DNAP treatment the observed band shift refers to the 8th nucleotide 3' to the recognition sequence 5'-GCAGC-3'.

DNA polymerases from Bacillus stearothermophilus, Bacillus caldotenax, and Bacillus caldovelox were purified by chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose and obtained in high yield. The enzyme preparations are free of exo- and endonuclease activities. Additional purification steps, e.g., hydrophobic interaction chromatography and chromatography on a Mono Q column or sucrose density gradient centrifugation, are needed to obtain the enzymes in the form of homogeneous 95-kDa proteins. Each of the three organisms possesses a major DNA polymerase activity comparable to DNA polymerase I. The enzymes require Mg2+ (10 to 30 mM) for optimal activity, although 0.4 mM Mn2+ could substitute for magnesium. The optimal reaction temperatures were lowest in B. stearothermophilus (60 to 65 degrees C) and about equal in B. caldovelox and B. caldotenax (65 to 70 degrees C). The thermal stabilities of the enzymes increased in the same order. The DNA polymerase from Thermus thermophilus was isolated for comparison by using a similar procedure. The enzyme was obtained as a homogeneous 85-kDa protein that was also free of exo- and endonucleolytic activities.

Small ribosomal subunits from rat liver have been studied by electron microscopy using freeze-drying and high-resolution shadow casting. The absolute hand of the asymmetric subunit has been determined and its three-dimensional model with a ‘right’ location of the side protuberance has been constructed. The results evidence that pro- and eukaryotic ribosomes have a unique and principally similar structural organization.

Expression of large T-antigen in COS cells can be inhibited by treatment of cell monolayers with oligodeoxyribonucleotides complementary to large T mRNA, which were covalently linked to poly-L-lysine. Strongest inhibition was observed with conjugates of oligodeoxynucleotides that hybridize to the sequence immediately 3' to the cap structure of the mRNA. Treatment of SV40 virus-infected CV-1 cells with the same conjugates reduces the virus-induced large T-antigen expression by more than 80%.

Native small ribosomal subunits (40SN) from rat liver and rabbit reticulocytes prepared at different KC1 concentrations have been investigated by electron microscopy after negative staining. Subunits of both origins show identical features. The initiation factor eIF-3 is located in the middle region of the convex rear side of the particles and covers an area extending from the protuberance at the interface up to the external surface. eIF-3 has the shape of a flat triangular prism and is attached with its triangular base to the ribosomal surface.

The 66 kDa protein present in a complexwith globin mRNA and 18 S rRNA [(1984) Eur. J. Biochem. 143, 27-33] has been reincorporated into functional eukaryotic initiation factor 3 (eIF-3) under conditions of protein synthesis. Additionally, two-dimensional polyacrylamide gel electrophoresis has been used to demonstrate the identity of the 66 kDa protein with the 66 kDa subunit ofeIF-3.

The three-dimensional structure of initiation factor eIF-3 and its binding site on native small ribosomal subunits have been analyzed by electron microscopic studies of native small ribosomal subunits and of initiation factor eIF-3 prepared from rat liver as well as by hydrodynamic studies of isolated eIF-3. Initiation factor eIF-3 has the shape of a flat triangular prism and is bound with its triangular base to the body part of the convex rear side of the small ribosomal subunit.

Native small ribosomal subunits from rabbit reticulocytes contain all initiation factors necessary for the formation of the mRNA-containing 48S pre-initiation complex. The complex formed in the presence of Met-tRNAf and 125I-labelled globin mRNA was cross-linked with diepoxybutane, and the covalent mRNA-protein complexes were isolated under denaturating conditions. The proteins of the covalent complex were identified as the 110, 95 and 66/64 kDa subunits of eIF-3. In addition, the 24 kDa cap binding protein and the ribosomal proteins S1, S3/3a, S6 and S11 were found covalently linked to the mRNA. Ribosomal proteins S3/3a and S6 were also involved in the ribosomal mRNA-binding domain of reticulocyte polysomes.

The binding site for eIF-3 on the small ribosomal subunit was studied (a) by use of a complex of eIF-3 and derived 40 S ribosomal subunit from rat liver, and (b) by use of native small ribosomal subunits from rabbit reticulocytes. After treatment of both complexes with dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate ribosomal proteins S3a, S4, S6, S7, S8, S9, S10, S23/24 and S27 became covalently linked to eIF-3 and were isolated together with the factor by gradient centrifugation. The ribosomal proteins were identified by two-dimensional polyacrylamide gel electrophoresis after periodate cleavage of the link(s).

The nearest neighbor relationships of major structural proteins within type D retroviruses (SMRV, MPMV, PMFV) were investigated by crosslinking with the cleavable bifunctional reagent dimethyl dithiobispropionimidate (DTBPI) or by use of 2-iminothiolane (methyl mercaptobutyrimidate, MBI) and subsequent oxidation by hydrogen peroxide. The crosslinked complexes of proteins were analyzed by two-dimensional diagonal polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In intact virions both crosslinking reagents induced the formation of covalently linked complexes of the internal structural proteins as well as complexes containing the envelope glycoproteins. The following complexes were found for each virus: SMRV: (pp20)2; (p35)2,4,6; (pp20-gp68); MPMV: (p10)2,4,6; (p25)2,4; (p10-p25); (gp20-gp68); (p45-gp68); PMFV: (p10)2; (p25)2; (p10-p25); (p45-gp68). In control experiments without crosslinking reagents no covalently linked complexes were detected.

Cross‐linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7‐dioxo‐5,6‐dihydroxy‐3,8‐diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion‐exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross‐link(s). The analysis revealed 42 cross‐linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross‐linked dimers implies that a large number of protein‐protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation.

The spatial relationships of major structural proteins within bovine leukemia virus (BLV) particles were investigated by crosslinking with the cleavable bifunctional reagent dimethyl dithiobispropionimidate (DTBPI) or methyl mercaptobutyrimidate (MBI). Covalently linked complexes of viral proteins were analyzed by two-dimensional diagonal polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both cross-linking reagents produced dimers of p10, p12, p15, and p24 as well as higher oligomers of p10 and p12. Furthermore three heterodimers, p10–p15, p10–p24, and gp30–gp64, were obtained. Surprisingly, BLV isolated in the absence of reducing agents already contained most of these complexes without use of any crosslinking reagents. This result demonstrates close proximities between sulfhydryl groups of certain structural proteins within BLV particles.

Initiation factor eIF-3 from rat liver forms a binary complex with the small ribosomal subunit. Within this complex, 18S ribosomal RNA can be cross-linked to the 66 000 dalton subunit of eIF-3 by treating the complex with a short bifunctional reagent, diepoxybutane, with a distance of 4A between the reactive groups. In binary complexes containing eIF-3 premodified with the heterobifunctional reagent, methyl-p-azido-benzoylaminoacetimidate (10A), the 66 000 dalton subunit of eIF-3 became covalently bound to 18S rRNA after irradiation of the complex with ultraviolet light. The involvement of only one of the eight eIF-3 subunits in the formation of the covalent RNA-protein complexes indicates a highly specific interaction between 18S rRNA and eIF-3 at the attachment site of the factor on the 40S subunit.

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25-40% saturation (0 degree C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation, eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3, sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.

In the quaternary initiation complex, eIF-2.GMPPCP.Met-tRNAf.40S ribosomal subunit, the Met-tRNAf can be cross-linked to the beta subunit of initiation factor eIF-2 as well as to ribosomal proteins S3a and S6 by treatment with the bifunctional reagent, diepoxybutane. Using 40S subunits, modified in advance with the heterobifunctional reagent, methyl-rho-azido-benzoylaminoacetimidate, Met-tRNAf is covalently bound to the same ribosomal proteins (S3a and S6) upon irradiation of the complex with ultraviolet light. Under both conditions proteins S3a and S6, together with a limited number of other ribosomal proteins, are covalently bound to 18S ribosomal RNA.