Conjunctive Fragment Research Articles

Total synthesis of horse heart cytochrome c

A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis 1,2, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis of an entire protein.

An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

Two-dimensional DNA fingerprinting of human individuals.

The limiting factor in the presently available techniques for the detection of DNA sequence variation in the human genome is the low resolution of Southern blot analysis. To increase the analytical power of this technique, we applied size fractionation of genomic DNA restriction fragments in conjunction with their sequence-dependent separation in denaturing gradient gels; the two-dimensional separation patterns obtained were subsequently transferred to nylon membranes. Hybridization analysis using minisatellite core sequences as probes resulted in two-dimensional genomic DNA fingerprints with a resolution of up to 625 separated spots per probe per human individual; by conventional Southern blot analysis, only 20-30 bands can be resolved. Using the two-dimensional DNA fingerprinting technique, we demonstrate in a small human pedigree the simultaneous transmission of 37 polymorphic fragments (out of 365 spots) for probe 33.15 and 105 polymorphic fragments (out of 625 spots) for probe 33.6. In addition, a mutation was detected in this pedigree by probe 33.6. We anticipate that this method will be of great use in studies aimed at (i) measuring human mutation frequencies, (ii) associating genetic variation with disease, (iii) analyzing genomic instability in relation to cancer and aging, and (iv) linkage analysis and mapping of disease genes.

Visualization of endogenous glycine in cat retina: an immunocytochemical study with Fab fragments

Fab fragments of a glycine antiserum were prepared and used for immunocytochemical visualization of glycine in the cat retina. The use of Fab fragments in conjunction with Fab-specific secondary and tertiary antisera improved tissue penetration and made it possible to identify a number of the immunoreactive neurons. Staining was observed in several subpopulations of amacrine cells, including the A7(AII) rod amacrine. Multiple subpopulations of cone bipolar cells were also seen to be immunoreactive. Many neurons exhibited no detectable immunostaining, indicating that general metabolic levels of glycine do not interfere with the visualization of those cells that contain large amounts of endogenous glycine. The distribution of immunostaining appears to parallel the pattern of glycine labeling seen previously with autoradiographic techniques and implicates these cells in glycine-mediated neurotransmission.

The EcoR124 and EcoR124/3 restriction and modification systems: Cloning the genes

The Esherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a “silent copy,” for the expression of a different R-M specificity R124/3. Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa. We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system. An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR 124/3 R-M system. These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity. Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities. Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function. Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally.

Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II.

The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.

Structure of tetanus toxin. The arrangement of papain digestion products within the heavy chain-light chain framework of extracellular toxin

Upon digestion with papain, single chain intracellular tetanus toxin was completely converted to the extracellular form of the toxin, which consists of two disulfide-linked polypeptide chains (heavy chain and light chain of tetanus toxin). A portion of the material was degraded further by papain to yield the two major protein fragments, B and C, respectively, previously identified after digesting extracellular tetanus toxin with this enzyme. Thus it may be concluded that the order of release of these fragments from the intracellular toxin does not provide a clue as to their position within the original molecule. However, N-terminal analysis of the two fragments in conjunction with recent N-terminal data on tetanus toxin itself clearly indicated that fragment B of tetanus toxin contains the light chain polypeptide and the N-terminal portion of the heavy chain, whereas fragment C is derived from the C-terminal portion of the heavy chain.

Studies on Immunosuppression by Cobra Venom Factor

Abstract CVF administered before immunization can profoundly depress humoral responses in C-sufficient mice. In AKR/J C5- mice given CVF before i.v. immunization with SRBC, only IgG levels were depressed, IgM titers being equivalent to those of untreated controls. The immunosuppressive effect became inapparent when the i.p. route of immunization was adopted. In DBA/2J C5- mice reduction of both IgG and IgM titers was observed irrespective of the route of immunization. The degree of suppression was, however, much more marked when mice were challenged intravenously. Essentially identical results were obtained with C5+ DBA/1J mice. These studies indicate that immunosuppression by CVF is unrelated to activation of the late C components. The significance of these findings is discussed with reference to the possibility that the generation of biologically active C fragments in conjunction with a C3 deficiency may account for immunosuppression by CVF.