Single chain antibody variable regions.

The use of antibodies or antibody fragments for targeting tumors (either for tumor imaging or as carriers for drugs or toxins), has encountered problems of clearance, and non-specific or inefficient binding in clinical trials. A novel approach, linking two antibody variable fragments (Fvs), with a short peptide to generate a continuous polypeptide chain, may be able to overcome some of these problems. Since these single chain antibody variable regions (scFvs), are transcribed from constructed 'genes', large-scale production in, for example, E. coli, should be straightforward.     

Analysis of non-polar regions in proteins.

A new method for finding hydrophobic nuclei and microclusters in protein structure is proposed. The method uses simple and clear-cut criteria based on an analysis of distances between the hydrocarbon groups of all residues. A detailed analysis of the composition and properties of hydrophobic nucleic and microclusters for proteins of different types has been carried out. This approach reveals that a hydrophobic nucleus can be composed not merely of classical hydrophobic amino acids, but also of dicarboxylic acids, their amides, arginine, lysine, histidine and tyrosine. The hydrophobic nucleus defined by this method should be considered as an individual structural unit along with such elements of the secondary structure as alpha-helices, beta-turns and beta-sheets.     

HLA-binding regions of HIV-1 proteins. I. Detection of seven HLA binding regions in the HIV-1 Nef protein.

The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.     

Retinoblastoma. A model of hereditary fragile chromosomal regions.

Direct karyotyping of tumour cells from three familial and two sporadic cases of retinoblastoma revealed the existence of a Dq- marker chromosome. The hypothesis is launched that a specific region on the long arm of one of the D chromosomes is the site of a locus which is essential for the sustained differentiation of specialized retinal tissue and may be the site of other loci essential for the maturation of other embryonic tissues. Fragility of this region and its potentiality for breakage under the influence of various environmental insults could be the basic cytological event leading to the development of sporadic retinoblastoma. Mutants at these loci, including those of sustained differentiation, could be a less common operational event whereby some variants could enhance the fragility of their respective chromosomal region and thereby explain the genetic transmission of retinoblastoma in certain families. It is common for the critical functional disruption of the locus to precede the deletion which may then be considered the terminal event in the fragile region.     

Conservation of chain reversal regions in proteins.

Using the bend frequencies based on 29 proteins in the previous paper (Chou and Fasman, 1979), beta-turn probability profiles were calculated for the C-peptides of 10 mammalian proinsulins, for 7 proteinase inhibitors, and for 12 species of pancreatic ribonucleases. Beta-turn correlation coefficient matrix tables were also computed to obtain the statistical mean between 45 pairs of proinsulin C-peptides, less than Ct greater than = 0.57 +/- 0.31; 21 pairs of proteinase inhibitors, less than Ct greater than = 0.73 +/- 0.13; and 66 pairs of ribonucleases, less than Ct greater than = 0.83 +/- 0.08. Despite relatively low sequence conservation in these three sets of proteins, beta-turns were predicted to be highly conserved: 33% sequence vs. 78% bend for the proinsulins, 20% sequence vs. 85% bend for the proteinase inhibitors, and 65% sequence vs. 92% bend for the ribonucleases. These results suggest that chain reversal regions play an essential role in keeping the active structural domains in hormones and enzymes intact for their specific biological function.     

Isolation of Azospirullum from diverse geographic regions.

We have isolated Azospirillum (Spirullum lipoferum) from roots of grasses of several genera collected from a number of tropical and subtropical-temperate locations. Pure cultures were obtained from a small percentage of samples; no higher percentage was secured from tropical than from other grasses. Acetylene reduction and distinctive growth in N-free soft agar deeps were inadequate to identify this genus, although helpful in initial screening. Fluorescent antibody tests with antiserum against characterized strains were helpful. There is some evidence that this genus of bacteria may be favored in the rhizoplane.     

Single-stranded regions on unintegrated avian retrovirus DNA.

Using chromatography on benzoylated naphthoylated DEAE-cellulose, we found that greater than 99.5% of the unintegrated linear viral DNA species detected in quail embryo cells infected with Rous sarcoma virus contained single-stranded regions, even at 16 h after infection. These regions were distributed across the genome and, on average, were primarily of plus-strand DNA. Within most of the linear viral DNA species, the minus strand was interpreted as being of genome size with two copies of the large terminal redundancy, LTR. In contrast, the plus strands in the linear viral DNA species were exclusively subgenomic.     

Multiple transforming regions of human cytomegalovirus DNA.

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Characterization of rat c-myc and adjacent regions.

Rat genomic regions covering c-myc were cloned from the DNA of both normal liver and two lines of Morris hepatomas, one of which had c-myc amplification. The three restriction maps showed perfect agreement within the overlapping regions. The 7 kb regions, which included the entire normal rat c-myc and the region 2.2 kb upstream, and one from the hepatomas, were sequenced and found to be identical. The coding regions of exons 2 and 3 were highly conserved between rat, mouse and man, but some differences in amino acids were noted. Exon 1 and the non-coding region of exon 3 showed limited homology between the three species. Rat exon 1 contained several nonsense codons in each frame and no ATG codon, indicating there to be no coding capacity in this exon. The 2.2 kb upstream regions and the introns compared showed unusual conservation between the rat and human genes. Some motifs, previously proposed as having a functional role in human c-myc, were also found in equivalent positions of the rat sequence. Nucleas S1 protection mapping revealed the second promoter to be preferentially used in most tissues or in hepatoma cells, and the second poly A addition signal to be the only one functional in all the RNA sources examined.