Eric Schabtach

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double‐strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD‐promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD‐chi site interactions in vivo we used phage lambdas terminase to introduce a site‐specific double‐strand break at lambdas cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi‐less DNA. Increased survival of chi‐containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi‐containing DNA molecules leads to RecA‐dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double‐strand break repair.

Structural analysis of membrane-bound retrovirus capsid proteins

We have developed a system for analysis of histidine‐tagged (His‐tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN‐containing monolayers specifically bound gold conjugates of His‐tagged proteins. Using PC+DHGN monolayers, we examined membrane‐bound arrays of an N‐terminal His‐tagged Moloney murine leukemia virus (M‐MuLV) capsid (CA) protein, His‐MoCA, and in vivo studies suggest that in vitro‐derived His‐MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His‐MoCA proteins formed extensive two‐dimensional (2D) protein crystals, with reflections out to 9.5 Å resolution. The image‐analyzed 2D projection of His‐MoCA arrays revealed a distinct cage‐like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein‐free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His‐tagged proteins.

The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin.

The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000, 40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.

The structure of hemoglobin from an unusual deep sea worm (Vestimentifera)

Abstract 1. 1. The extracellular hemoglobin from a species of Vestimentifera, obtained from hydrothermal vents at 2.5 km depths of the ocean, has been studied. 2. 2. The hemoglobin molecules appear as two-tiered hexagonal structures in the electron microscope. The hexagons measure 160 A high and 245 A wide. 3. 3. The pigment, which was purified from frozen specimens, appears to be very unstable and elutes on Sepharose 4B as aggregates of approx 1.7 × 10 6 and 400,000 mol. wt. 4. 4. The subunit molecular weight of purified hemoglobin, determined by sodium dodecyl sulfate gel electrophoresis, shows heterogeneity with molecular weights around 15,000 and 30,000. 5. 5. The purified hemoglobin contains one heme per 23,000 g protein. The amino acid compositions of isolated fractions are presented. 6. 6. This Vestimentifera, which shows characteristics of both the phylum Pogonophora as well as Annelida, has an extracellular hemoglobin whose structure is very similar to that of annelid extracellular hemoglobins.

Comparison of chlorocruorin and annelid hemoglobin quaternary structures

Abstract 1. 1. The structures of Eudistylia vancouveri chlorocruorin and Pista pacifica and Thelepus crispus extracellular hemoglobins have been examined by electron microscopy. The intact pigments display a two-tiered hexagonal array characteristic of other annelid high molecular weight hemoglobins and chlorocruorins. 2. 2. The smallest subunit molecular weight of these pigments determined by SDS gel electrophoresis is 14–15,000; Pista hemoglobin contains some additional 30,000 molecular weight material under a variety of denaturing conditions. Earthworm hemoglobin is also heterogenous upon SDS gel electrophoresis, consisting of at least six components with mol. wt ranging from 13, 000 to 35,000. 3. 3. The amino acid compositions of these high molecular weight pigments are compared with one another and with the intracellular hemoglobins of other invertebrates and vertebrates. It is likely that Eudistylia chlorocruorin and certain high molecular weight annelid hemoglobins form a group of closely related proteins.

Erythrocruorins of Euzonus mucronata treadwell: Evidence for a dimeric annelid extracellular hemoglobin

Abstract 1. 1. The extracellular hemoglobin of the opheliid polychaete Euzonus mucronata is unusual in that it consists of a 3.2 × 10 6 as well as a 6–7 × 10 6 molecular weight hemoglobin at pH 7.O. 2. 2. Electron microscopy of the 3.2 x 10 6 dalton material shows the typical annelid hemoglobin pattern of a two-tiered hexagonal array of submultiples whereas the 6–7 × 10 6 dalton pigment has a four-tiered hexagonal arrangement, a putative dimer of a 3.2 × 10 6 molecular weight material. A few six-tiered trimeric stacks are also present. 3. 3. The dimeric material, which represents 10–15% of the total pigment, is present in unpurified vascular fluid as well as in purified hemoglobin. The constant stoichiometry between the 3.2 × 10 6 dalton monomer and the dimeric pigment under different methods of purification as well as in hemoglobin examined directly from the worm suggests that the dimer is not a preparative artifact. The dimer is not in a state of rapid dissociation-association equilibrium with the monomer nor does the dimer seem to be the result of a ligand-linked association of the 3.2 × 10 6 dalton monomer. 4. 4. The subunit molecular weights of both the monomer and dimer are the same with polypeptide chains of 36,700 ± 800, 31,400 ± 800. 14,400 ± 300. and 12,500 ± 300 as determined by sodium dodecyl sulfate gel electrophoresis. The same molecular weight heterogeneity is present for globin which has been performic acid oxidized, carboxymethylated in 7–8 M guanidine HC1 and 0.1 M in 2-mercaptoethanol, or treated with 8 M urea, 0.1 M in 2-mercaptoethanol. 5. 5. The hemoglobin contains one heme per 22,400 g protein. The amino acid composition of this protein is presented.

Assembly of Octopus dofleini hemocyanin : a study of the kinetics by sedimentation, light scattering and electron microscopy

The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared.

Developmental interactions in the growth and branching of the lateral dendrite of Mauthner's cell (Ambystoma mexicanum).

Abstract The axolotl Mauthner (M) cell receives synapses from the vestibular and lateral line nerves on highly branched regions located ventrally and dorsally, respectively, of its lateral dendrite. One of the pair of M-cells was deprived of all ipsilateral vestibular supply and of some lateral line supply by unilateral ablation of the otic vesicle at stage 34, before nerve outgrowth and M-cell differentiation. Histological reconstruction of such deprived M-cells at stages 42 and 45, following M-cell differentiation, revealed that the deprived dendrite was poorly developed, being thinner and much less ramified than in controls. This effect was specific; no changes were consistently observed in the shape or size of the M-cell body, the medial dendrite, or the axon. Electron microscopic examination of deprived M-cells showed that morphologically normal synapses were present on the lateral dendrite; however, synaptic knobs identifiable as being of vestibular origin were absent. We suggest that patterned growth and branching of the M-lateral dendrite during differentiation is regulated through interactions with afferent axons.

Structural and functional properties of hemoglobin from the vestimentiferan Pogonophora, Lamellibrachia

Representatives of the phylum Pogonophora were found at 1800 m in a fault region off San Diego, CA, where there were indications of recently discharged hydrothermal fluids. The worm-like organisms were determined to be Lamellibrachia sp, phylogenetically related to Riftia pachyptila, a Pogonophora from the Galapagos Rift hydrothermal vent site whose hemoglobins were previously studied. Lamellibrachia extracellular hemoglobin was found to be heterogeneous with respect to its state of aggregation. Two tiered hexagonal structures, as described for annelid hemoglobin, with molecular weights of approx. 3 · 106, make up 25–40% of the hemoglobin, while the other hemoglobin fraction has a molecular weight of 300 000–400 000. The two molecular weight fractions have different subunit compositions and are not in equilibrium with one another. Their amino acid compositions differ slightly. Both fractions contain 1 mol of heme per approx. 25 000 g of protein. The unfractionated hemoglobin has a high oxygen affinity (P50 = 0.16 Torr, 20°C) and little or no pH sensitivity, and shows cooperative subunit interactions (nH = 2–2.7). Lamellibrachia hemoglobin closely resembles the extracellular hemoglobins of annelids in many respects but shares the molecular weight heterogeneity previously reported for hemoglobin of R. pachyptila.

Chiton hemocyanin structure

Abstract 1. 1. Hemocyanin from the chiton, Katharina tunicata , has a sedimentation coefficient (S o 20.w ) of 61.2S, M r = 4.2 × 10 6 , at pH 7.0 in the presence of 10 mM MgCl 2 . 2. 2. In electron micrographs, the 61S hemocyanin appears as a three-tiered cylinder, 31 nm in dia. and 19 nm in length, with a polar profile. 3. 3. An increase in alkalinity results in gradual dissociation to 20S and 11S components, which correspond to one-fifth and one-tenth molecules, respectively. The 61S molecule is stabilized in buffers containing MgCl 2 . 4. 4. SDS and regular gel electrophoresis of K. tunicata hemocyanin show a single subunit with an apparent M r of about 350,000 or greater. 5. 5. Although the 11S subunits are homogeneous, digestion studies indicate that the functional domains are heterogeneous. 6. 6. The hemocyanins from each molluscan class, chiton, cephalopod and gastropod, appear to be distinctive.