Ekkehard K. F. Bautz

Developmental regulation of a constitutively expressed mouse mRNA encoding a 72-kDa heat shock-like protein

Multiple heat shock cognate (hsc70) cDNA clones were isolated from the mouse embryonal carcinoma cell line F9. They all encode a single 72-kDa protein, which is constitutively expressed in all mouse cell lines and tissues tested, and which is only slightly induced by hyperthermia. hsc70 RNA is very abundant in F9 stem cells and brain, but very little is found in 14-day-old embryos. Upon differentiation of F9 stem cells induced by retinoic acid and cyclic AMP, expression of the hsc70 gene decreases only slightly, suggesting that hsc70 is highly expressed in early mouse development and is then down-regulated towards the end of embryogenesis. In adult tissues only the brain retains the high level of hsc70 gene expression found in F9 stem cells. We also show that expression of hsc70 protein and clathrin is uncoupled in F9 cells, indicating that the uncoating activity of coated vesicles may not be the only function of hsc70 protein.

Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster

SummaryMonoclonal antibodies were raised against purified RNA polymerase II (or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distint regions of RNA polymerase activity in this puff.

Baculovirus expression cassette vectors for rapid production of complete human IgG from phage display selected antibody fragments.

For the expression of human intact IgG antibodies, we have constructed a set of baculovirus expression vectors designed to facilitate rapid insertion of heavy and light chain genes of Fab or scFv antibodies derived from phage display antibody libraries. By linking them to human constant or Fc regions, expression of complete human immunoglobulin molecules was achieved in insect cells by infection with recombinant baculovirus. The IgG expression cassette vectors are based on the backbone vector which contains two back to back polyhedron and p10 promoters. The IgG expression cassette elements, including the authentic IgG lambda or kappa and heavy chain signal sequences, as well as light chain (lambda or kappa) and heavy chain constant region genes are combined in a single vector and are controlled by the p10 and polyhedron promoter respectively. Either of VL or Fab-L and VH or Fab-Fd genes from common phage display systems can be directly inserted into one of the cassette vectors through in-frame cloning sites. This design of a single cassette vector combining heavy and light chain expression elements allowed rapid production and secretion of correctly processed and assembled intact immunoglobulins from recombinant baculovirus infected insect cells. The recombinant antibodies showed the expected molecular size of the H2L2 heterodimer in non reducing SDS-PAGE. No apparent differences were found between the expression level of heavy and light chains, and antigen binding function was preserved. For various antibodies, yields between 6 and 18 mg/l IgG were obtained.

A Major Antigenic Domain of Hantaviruses is Located on the Aminoproximal Site of the Viral Nucleocapsid Protein

Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli. The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins. This fragment was recognized by all HFRS patient sera tested (n=128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.

Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence.

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.

MAPPING OF LINEAR EPITOPES RECOGNIZED BY MONOCLONAL ANTIBODIES WITH GENE-FRAGMENT PHAGE DISPLAY LIBRARIES

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5′ terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

Fine mapping of the antigen–antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site‐specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha‐helical structure for the main contact area. Copyright

Heat-shock proteins are associated with hnRNA in Drosophila melanogaster tissue culture cells

Ribonucleoprotein complexes of Drosophila melanogaster Kc tissue culture cells grown at 24°C or heat‐shocked at 37°C were cross‐linked in vivo by u.v. irradiation. Cross‐linked heterogeneous nuclear ribonucleoprotein (hnRNP) complexes were fractionated by oligo(dT)‐cellulose chromatography and CsCI density centrifugation. The hnRNP complexes of both 24°C and 37°C culture cells possess buoyant densities in CsCI between ϱ = 1.38 g/cm‐3 and 1.43 g/cm‐3. The 35S‐labelled proteins bound to the hnRNA of 37°C culture cells correspond in mol. wt. to the so‐called heat‐shock proteins of 70 K, 68 K, 27 K, 26 K, 23 K and 22 K. The 70 K and 68 K proteins are also present in hnRNP complexes of 24°C culture cells. In addition, several other Drosophila hnRNPs of 140 K, 56 K, 45 K, 43 K, 38 K, 37 K and 34 K, whose synthesis is strongly repressed under heat‐shock conditions, could be identified. The results demonstrate that the so‐called heat‐shock proteins possess a function as RNPs.

Heat-shock puff 93 D from Drosophila melanogaster: accumulation of a RNP-specific antigen associated with giant particles of possible storage function.

The monoclonal antibody P11 is directed against a 38 000 dalton protein of Drosophila melanogaster. On polytene chromosomes this protein is present in a subset of the RNA polymerase II‐containing loci. Here we show by density centrifugation and enzyme‐linked immunosorbent assay tests that the P11 antigen is part of nuclear ribonucleoprotein (RNP) complexes. Indirect immunofluorescence shows that, after prolonged heat‐shock, the P11 antigen is present only in the heat‐shock puff 93 D. Identical distribution patterns were obtained with another monoclonal antibody, Q18. Unlike P11, this antibody also cross‐reacts with D. hydei and D. virilis polytene chromosomes, where the puffs 48 B and 20 CD, respectively, are the only loci prominently stained after heat‐shock. The small and giant RNP complexes previously described in these puffs were also observed in puff 93 D. Both types of particle contain the P11 antigen as shown by immunoelectron microscopy. We suggest that the P11 antigen is associated with a special class of RNPs which are possibly involved in the storage of primary transcription products inside the nucleus.

Human recombinant neutralizing antibodies against hantaan virus G2 protein

Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients.