Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the -centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical -or -centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the -centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the -centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus -centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the -centredtrp operator shows a change in the specificity of binding to a -centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the -centredtrp operator.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Genetic polymorphisms of gene conversion within the duplicated human α-globin loci

Summary Of the 645 Japanese subjects studied, we have identified 10 individuals heterozygous for a chromosome with the triplicated -globin loci. The frequency of the triple -loci was 0.008 in this population, while that of the single -locus, i.e., -thalassemia 2 gene, might be lower than 0.0008. Analysis of haplotypes using particular RsaI site polymorphism in the -globin gene complex strongly suggests that the triple loci may have had multiple origins in this population.     

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

Summary The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, -, -, and -crystallins, and each class represents a multigene family. The -crystallin gene family consists of 1-crystallin (CRYA1) and 2-crystallin (CRYA2) genes (previously designated A-and B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human 2-crystallin gene and does not cross-hybridize to 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.     

Requirement of the newly recognized ilvJ gene for the expression of a valine transaminase activity in E. coli K-12

Summary The isoleucine--ketoglutarate and valine--ketoglutarate transaminase activities have been attributed to an enzyme coded in E. coli K-12 by the ilvE gene. I report here evidence that these two activities can be dissociated and appear to be the products of two different genes. Mutants altered in the ilvE gene are devoid of isoleucine--ketoglutarate transaminase activity and possess a normal valine--ketoglutarate transaminase activity. I describe here mutants lacking valine transaminase activity. They are altered in a gene, ilvJ, located between ilvE and ilvD at 83 min on the E. coli K-12 map. Temperature-sensitive revertants of the mutant containing the ilvJ mutation show a temperature-sensitive valine--ketoglutarate transaminase activity. I conclude that ilvJ is the structural gene for valine--ketoglutarate transaminase.     

Elongation factor 1α genes of the red alga Porphyra purpurea include a novel, developmentally specialized variant

The life cycle of the red alga Porphyra purpurea alternates between two morphologically distinct phases: a shell-boring, filamentous sporophyte and a free-living, foliose gametophyte. From a subtracted cDNA library enriched for sporophyte-specific sequences, we isolated a cDNA encoding an unusual elongation factor 1 (EF-1) that is expressed only in the sporophyte. A second EF-1 gene that is expressed equally in the sporophyte and the gametophyte was isolated from a genomic library. These are the only EF-1 genes detectable in P. purpurea. The constitutively expressed gene encodes and EF-1 very similar to those of most eukaryotes. However, the sporophyte-specific EF-1 is one of the most divergent yet described, with nine insertions or deletions ranging in size from 1 to 26 amino acids. This is the first report of a developmental stage-specific EF-1 outside of the animal kingdom and suggests a fundamental role for EF-1 in the developmental process.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Syntheses of alpha-dystroglycan derived glycosyl amino acids carrying a novel mannosyl serine/threonine linkage

-Dystroglycan (-DG) is a membrane-associated, extracellular glycoprotein. It is anchored to the cell-membrane by binding to the transmembrane glycoprotein -dystroglycan (-DG) to form an /-DG-complex. It was discovered that the bovine peripheral nerve -DG possesses the Ser/Thr linked tetrasaccharide as the major constituent of the O-linked carbohydrates, which was proposed to contribute laminin binding activity of this glycoprotein.This structure has a striking feature in terms of the mode of linkage between oligosaccharide and the core protein. It has a mannose residue linked to the core protein through Ser/Thr residue. A similar structure was proposed to exist in brain derived HNK-1 immunoreactive O-glycans. Being interested in the structural novelty and potential biological significance of this type of glycan chains, the chemical synthesis of Ser/Thr linked mannose containing tetrasaccharide was investigated. Tetrasaccharide donor was constructed from monosaccharide blocks and coupled with Ser/Thr derivatives. Subsequent deprotection afforded target tetraosyl serine. Furthermore, synthetic routes to lower homologues, namely Gal--(1,4)-GlcNAc--(1,2)-Man--Ser and GlcNAc--(1,2)-Man--Ser were also provided.     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Haemoglobin synthesis in 28 obligatory cases for α-thalassemia traits

Summary In the Far East two types of -thalassemia genes, namely -thalassemia₁ (-thal₁) and -thalassemia₂ (-thal₂) exist. Definite diagnosis of the -thal₁ and -thal₂ traits is very difficult because their hematological findings are minimally abnormal or normal. This study attempts to characterize the heterozygotes by hemoglobin chain synthesis in reticulocytes from obligatory cases of the -thal₁ and -thal₂ traits. Twelve parents of babies with hemoglobin Bart's hydrops fetalis (obligatory -thal₁ trait) had the mean total radioactivity / ratio of 0.76±SD 0.04, while that of 7 normal controls was 1.06±SD 0.04. The / globin chain ratios of 16 cases, who were either parents or offspring of patients with hemoglobin H disease, were found to segregate into 2 groups, i.e. 0.78±SD 0.03 (10 cases) and 0.92±SD 0.03 (6 cases), probably representing the -thal₁ and -thal₂ traits respectively. The hematological data of the first group showed definite hypochromic microcytic red cells, similar to thoseof the parents of the hydrops. The second group had significantly higher mean corpuscular hemoglobin than the first group, compatible with -thal₂ trait. Our globin chain synthesis study thus appears to be capable of discriminating normal, -thal₁ and -thal₂ traits.A preliminary report of the results was presented at the XV Congress of the International Society of Haematology, Israel, September, 1974.     

Over expression of integrin α 5 β 1 in human hepatocellular carcinoma cell line suppresses cell proliferation in vitro and tumorigenicity in nude mice

Integrin 5 1 and 2 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin 5 1 was decreased and another integrin 6 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin 5 and/or 1 cDNAs were established, and designated 5 1.6-7721, 5.3-7721, and 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin 5 and 1 subunits resulted in the overexpression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in 5.3-7721, 1.6-7721, 5 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with 1.6-7721,and 5 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin 5 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin 5 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.     

Evidence that expression of SpαI/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous α-spectrin allele

Summary Many cases of hereditary elliptocytosis (HE) result from mutated spectrin -chains. It has repeatedly been observed that the amount of a mutant -chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp^I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp^I/65 variant, suggesting the existence, in trans, of a low percentage -allele. The -spectrin gene haplotype associated with the latter was constantly – +–, based on the XbaI, PvuII and MspI polymorphic sites. In contrast, a basal level of expression of the Sp^I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage -allele. The haplotype corresponding to this other -allele was + – +. Study of another generation of the family showed, however, that the – + – haplotype could also be linked to a normal percentage -allele. These results are consistent with the view that the expression level of ^I/65 spectrin (and of other types of -variants) is compounded by a genetic factor that is linked to the normal -allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.     

Testosterone and progesterone metabolism in the central nervous system: Cellular localization and mechanism of control of the enzymes involved

Summary This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia.1. The activities of 5-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5-androstane-3,17-diol; 3-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5-reductase activity. A completely different localization was observed for 3-hydroxysteroid dehydrogenase; the formation of 3-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5-reductase and 3-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5-reductase, which metabolizes progesterone into 5-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-reduce progesterone. On the contrary, 3-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5-pregnane-3-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.     

Interleukin-1α: Its possible roles in cancer therapy

Our studies on recombinant human IL-1 polypeptide were summarized with respect to molecular cloning, production, quantitative assay systems, antitumor activity, myelorestorative activity and augmentation of host resistance to infections.Recombinant human IL-1 (18 kDa) was produced through the expression of the cloned human IL-1 cDNA inEscherichia coli and purified to an endotoxin-free homogeneous polypeptide. The human IL-1 inhibited dose-dependently the growth of syngeneic murine tumors transplanted in mice and completely regressed the tumors in some cases, and its antitumor activity was significantly enhanced in combination with indomethacin. The human IL-1 accelerated the recovery of the numbers of peripheral leukocytes and neutrophils in a dose-dependent manner at a dose as low as 10 ng/mouse/day in myelo suppressed mouse model produced by administering anticancer chemotherapeutic drugs. The myelorestorative effect of IL-1 was observed not only on leukocytes/neutrophils, but also on platelets in myelosuppressed mice. In addition, the human IL-1 markedly augmented dose-dependently resistance of normal and leukopenic mice to various microbial infections.These results suggested that recombinant human IL-1 might be useful for cancer therapy from the viewpoints of improving adverse effects such as myelosuppression caused by chemotherapy and/or radiation therapy and preventing infections. In addition, use of IL-1 may permit more intensive chemo- and radiation therapies using higher doses. Finally, the antitumor activity of the IL-1 itself may play an important role.     

A note on dimensionless life histories for birds versus mammals

Summary Offspring production over the adult lifespan (b/M whereb is the yearly fledgling or offspring production and 1/M is the mean adult lifespan) is an approximate invariant within both birds and mammals. The two taxa differ, however, in that mammals have bothM and b as invariants (b/M = b/M) while birds do not ( is the age at first breeding). Birds have a surprising cancellation in that bothM andb are ^–0.25.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Control of formation of active soluble or inactive insoluble baker''s yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

Summary Using standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast -glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated -glucosidase granules in Escherichia coli. Under these conditions active soluble -glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active -glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacI ^qrepressor gene on an R-plasmid. The formation of active soluble -glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - RB refractile body - t-PA tissue type plasminogen activator - IFN interferon - X--glucoside 5-bromo-4-chloro-indolyl--D-glucopyranoside