Showing papers by "Laboratory of Molecular Biology published in 1985"

Hydrogen bonding and biological specificity analysed by protein engineering

Abstract: The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5–1.5 kcal mol−1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further ∼3 kcal mol−1.

Isolation and genetic characterization of human KB cell lines resistant to multiple drugs.

Abstract: Human KB cell lines resistant to high levels of colchicine were isolated by several successive single-step selections. Most of these selection steps resulted in cross-resistance to vincristine, vinblastine, adriamycin, actinomycin D, and puromycin; however, at the highest levels of colchicine resistance, increased cross-resistance to other drugs was not observed. There was no major change in protein synthesis or alteration in protein phosphorylation or [14C]glucosamine labeling patterns accompanying the development of multiple drug resistance as measured by analysis of metabolically labeled proteins on SDS gels. Cell-cell hybridization experiments showed that the colchicine-resistant and multiple drug-resistant phenotypes were incompletely dominant. In addition, colchicine resistance was found to segregate independently from resistance to other drugs in one somatic cell hybrid, suggesting that complex genetic loci are involved in the development of the multiple drug-resistant phenotype. These mutants should be useful for the study of the clinically important problem of multiple drug resistance in human cancer.

β -Hairpin families in globular proteins

Abstract: Beta-hairpins, one of the simplest supersecondary structures, are widespread in globular proteins, and have often been suggested as possible sites for nucleation. Here we consider the conformation and sequences of the loop regions of beta-hairpins by analysing proteins of known structure. We find that the 'tight' beta-hairpins, classified by the length and conformations of their loop regions, form distinct families and that the loop regions of the family members have sequences which are characteristic of that family. The two-residue hairpin loops include almost entirely I' or II' beta-turns, in contrast to the general preference for type I and type II turns. These findings are being used to help define templates or consensus sequences to be incorporated into our existing supersecondary structure prediction algorithm. This information can also be used in model-building homologous proteins.

The Origin and Evolution of Retroposons

Abstract: Publisher Summary This chapter describes the origin and evolution of retroposons. It deals with two of them which operate on very different timescales: (1) RNA splicing (particularly messenger RNA splicing), which is the excision of transcribed noncoding sequences from RNA during normal gene expression and (2) retroposition, which is the insertion of reverse-transcribed sequences from RNA back into the genome during evolution. RNA splicing was the first and most surprising discovery of eukaryotic molecular genetics. Retroposons are a class of dispersed sequences in DNA, which appear to have arisen during evolution by a particular mechanism of inserting RNA sequences into chromosomal DNA (retroposition). They include processed mRNA pseudogenes, snRNA pseudogenes, the highly repeated ALU sequences, and a variety of other sequences. They are entirely distinct from transposons and retroviruses. The chapter explains the splicing of ribosomal RNA, mitochondrial RNA, and chloroplast RNA.

Parasegments and compartments in the Drosophila embryo

Abstract: One of the first steps in segmentation of the Drosophila embryo seems to be not the formation of segments, but instead the definition of 14 domains, each of which encroaches into adjacent segments. We call these domains parasegments and discuss their developmental significance.

Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins.

Abstract: The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.

A hapten-specific chimaeric IgE antibody with human physiological effector function.

Abstract: Immunoglobulin E (IgE; see ref. 1) has a central role in allergic reactions although it rarely exceeds 5 µg ml−1 even in the serum of severely allergic individuals. Both mast cells and basophils possess receptors which bind the Fc portion of IgE with high affinity; crosslinking of membrane-bound IgE by allergen results in degranulation of the cell and release of a variety of pharmacologically active mediators including histamine. Myeloma IgE has been successfully used to block the skin sensitizing activity of allergic sera2; however, human myeloma IgE is clearly in limited supply. The emergence of techniques allowing the stable introduction of immunoglobulin gene DNA into myeloma cells3–5 has allowed us to construct a mouse cell line that secretes a chimaeric IgE, λ1 antibody whose heavy chain is composed of a human Ce constant region fused to a mouse variable (VH) region. This chimaeric IgE is specific for the hapten 4-hydroxy-3-nitro-phenacetyl (NP) and can, when crosslinked by antigen, trigger the degranulation of human basophils. When not crosslinked, however, the chimaeric IgE can prevent the passive sensitization of these cells by sera from allergic subjects.

Two tandemly organized human genes encoding the T-cell gamma constant-region sequences show multiple rearrangement in different T-cell types.

Abstract: The recent detailed analysis of genes that undergo rearrangement in T cells has shown that the T-cell receptor genes encoding alpha- and beta-chains are involved in specific alterations in T-cell DNA analogous to the immunoglobulin genes. A third type of gene, designated gamma, has been isolated from mouse cytotoxic T lymphocytes, and evidence suggest that the mouse displays very limited diversity in this gene system, having only three variable-region (V) genes and three constant-region (C) genes. The function of the so-called T-cell gamma gene is unknown. We have isolated genomic genes encoding the human homologue of the mouse T-cell gamma gene; as there is no evidence that this T-cell rearranging gene is anything to do with the T3 molecule, we have designated the human T-cell rearranging gene as TRG gamma (ref. 13), to avoid confusion with the T3 gamma-chain, and have shown that the gene locus maps to chromosome 7 in humans. We now report that human DNA contains two tandemly arranged TRG gamma constant-region genes about 16 kilobases apart. These two genes show multiple rearrangement patterns in a variety of T cells, including helper and cytotoxic/suppressor type, as well as in all forms of T-cell leukaemia. Our results indicate variability of this T-cell gene system in man compared with the analogous system in mouse.

Molecular events during maturation of the immune response to oxazolone.

Abstract: Sequence analysis of the heavy- and light-chain messenger RNA of hybridomas immunized with a specific hapten yields important clues about the interplay between genetic and selective events during the onset and maturation of the immune response. The maturation of the primary response to the hapten 2-phenyl-5-oxazolone is characterized by a drift to higher-affinity somatic variants of a germline-encoded basic sequence, whereas hybridomas from the secondary response demonstrate a further maturation dominated by a shift to alternative germline combinations.

Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis.

Abstract: An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor.

Abstract: U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.

Crossbridge behaviour during muscle contraction

Abstract: A number of recent observations by probe and X-ray methods on the behaviour of crossbridges during contraction is considered in relation to the energetics of the process. It is shown that a self-consistent picture of the crossbridge cycle, compatible with these observations and involving strongly and weakly attached crossbridges, can be obtained providing that the tension-generating part of the crossbridge stroke is only about 40 A i.e. about one-third of the usually accepted value. The myosin head subunits in the tension-generating bridges could have a configuration close to that of rigor. A mechanism is suggested whereby rapid tension recovery after quick releases up to 120 A could still be produced by such a system.

The distribution of Ultrabithorax transcripts in Drosophila embryos.

Abstract: We have used in situ hybridization to monitor the distribution of Ultrabithorax (Ubx) transcripts during the early stages of Drosophila embryogenesis. When first detectable, in the late syncytial blastoderm, Ubx transcripts are broadly distributed from 20 to 50% egg length (measured from the posterior pole). By the completion of cellular blastoderm formation, a precisely bounded zone one segment wide is defined by the accumulation of high levels of Ubx transcripts. This zone is probably the primordium for parasegment 6, that is, the posterior compartment of the third thoracic segment (T3) and the anterior compartment of the first abdominal segment (A1). Following gastrulation, seven metameric units of the germ band express Ubx transcripts in most or all cells of both the ectoderm and the mesoderm. This principal domain of Ubx expression is sharply bounded at superficial grooves in the embryo which probably mark the A/P compartment boundaries in T3 and A7, and so consists of parasegments 6-12. Outside this region, Ubx transcripts can be detected in only a few cells of the ectoderm of parasegments 5 and 13, and in the amnioserosa. This distribution suggests that the major role of Ubx in the early embryo lies in the morphological domain generally ascribed to the control of the bithoraxoid gene. Later in embryogenesis, the pattern of Ubx transcript accumulation is different in each of the major germ layers, suggesting that different controls regulate Ubx in each. In the ectoderm, but not in the mesoderm, Ubx transcripts accumulate differentially in anterior and posterior compartments. Probes prepared from different regions of the Ubx transcription unit show related but different patterns of hybridization. These patterns suggest that the 3.2-kb Ubx transcript is distributed throughout the wider domain of Ubx expression, but that the 4.7-kb transcript accumulates preferentially in parasegment 6.