Sequence analysis reveals a beta-thalassaemia mutation in the DNA of skeletal remains from the archaeological site of Akhziv, Israel.

beta-Thalassaemia is manifested by severe anaemia and extensive bone pathology. Similar pathology may also result from other forms of anaemia. To clarify the precise cause, we performed DNA analyses on archaeological remains of a child with severe bone pathology. We found homozygosity for frameshift in codon 8 of beta-globin, causing a beta-null phenotype. Paradoxically, the child died when eight years old, whereas such patients are transfusion dependent from early infancy. An infrequent polymorphic marker in the child's DNA, and information from present-day patients, indicated that amelioration of the clinical condition was due to elevated fetal haemoglobin production. Thus this analysis provided not only precise diagnosis of a genetic disease but also allowed clarification of the molecular mechanism underlying the clinical presentation.

Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.

Crystal structure of a bacterial chitinase at 2.3 A resolution.

BACKGROUND: Chitinases cleave the beta-1-4-glycosidic bond between the N-acetyl-D-glucosamine units of which chitin is comprised. Chitinases are present in plants, bacteria and fungi, but whereas structures are available for two prototypic plant enzymes, no structure is available for a bacterial or fungal chitinase. RESULTS: To redress this imbalance, the structure of native chitinase A from Serratia marcescens has been solved by multiple isomorphous replacement and refined at 2.3 A resolution, resulting in a crystallographic R-factor of 16.2%. The enzyme comprises three domains: an all beta-strand amino-terminal domain, a catalytic alpha/beta-barrel domain, and a small alpha+beta-fold domain. There are several residues with unusual geometries in the structure. Structure determination of chitinase A in complex with N,N',N",N"'-tetra-acetylo-chitotetraose, together with biochemical and sequence analysis data, enabled the positions of the active-site and catalytic residues to be proposed. CONCLUSIONS: The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis. The role of the amino-terminal domain could not be identified, but it has similarities to the fibronectin III domain. This domain may possibly facilitate the interaction of chitinase A with chitin.

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

We found that the activity of the phage lambda PL promoter is inversely dependent on temperature. Both in vivo and in vitro transcription assays revealed that the rather unique temperature response of PL is a sum of an intrinsic property of the promoter and its activation by integration host factor. We also found that at low temperature, phage lambda can lysogenize efficiently but cannot complete the lytic cycle. We hypothesize that by sensing the low environmental temperature the PL promoter plays a role in determining the direction of phage lambda development.

The SV40 capsid protein VP3 cooperates with the cellular transcription factor Sp1 in DNA-binding and in regulating viral promoter activity.

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Dynamics of the nucleoprotein structure of simian virus 40 regulatory region during viral development.

The regulatory region of SV40 is composed of multiple elements, including the origin of replication (ori), the encapsidation signal (ses) and the enhancer. Here, the structure of the chromatin and nucleoprotein complexes in a region encompassing ses and part of the enhancer was investigated in detail by in situ probing with DNase I. We have used a model experimental system based on plasmids which carry parts of the SV40 regulatory region. The results demonstrate that a specific nucleoprotein structure at the region is formed early after transfection. The overall structure is maintained throughout the viral life cycle. The observed DNase digestion pattern is consistent with the presence of a mixed population of viral minichromosomes with various, but not random, nucleosomal arrangements in that region. Specific modulations, which are associated with the various stages of the viral life cycle, are superimposed on the general structure. The most dramatic changes occur at nucleotides 34 and 113, located at both ends of ses and flanking the GC-box region. Some of the changes depend on the presence of viral gene product(s), probably a late (capsid) protein. The results further suggest that the condensed minichromosome within the viral particle assumes a highly specific configuration in this region. The nucleoprotein structure is sensitive to modifications of the primary nucleotide sequence and to flanking DNA elements. There is good correlation between distortions in the nucleoprotein structure and the inability of mutant plasmids to be packaged, substantiating the requirement for proper chromatin condensation in viral packaging.

Repression of the lambda pcin promoter by integrative host factor.

The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda. The pcin promoter transcribes the cI repressor gene constitutively. lambda cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF). lambda cI- cin-1 grows normally in IHF- mutants, indicating that repressor overproduction from pcin blocks lytic growth. The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription. We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells. In vitro studies show that IHF directly inhibits transcription initiation at pcin. Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter. RNA polymerase bound in an open promoter complex is resistant to IHF. We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure.

Deletion analysis of the retroregulatory site for the lambda int gene.

The lambda int gene product, integrase, recombines phage and bacterial DNA at a specific site during the integration step of lysogeny. Regulation of integrase synthesis is complex. (1) Transcription of the gene can occur from either of two promoters. lambda cII protein activates transcription initiation near int at pI. The lambda N protein allows transcription of int from pL. N protein acts by preventing transcription termination at several terminators between pL and int. (2) The expression of integrase is also subject to post-transcriptional regulation by a site, sib, which is located beyond int in the b region of lambda. Expression of int from pL is inhibited by sib, whereas that from pI is not. The negative control of int expression by sib is termed retroregulation. Retroregulation of int is caused, in part, by processing of the pL transcript at the sib site by RNase III of Escherichia coli. The exonuclease, Bal31, was used to generate a set of deletions to define the sib regulatory site. Both sib+ and sib- deletions were sequenced, and it was concluded from this and other work that a dyad symmetry present in the b region, 270 base-pairs from int, was necessary for retroregulation. The RNA structure of this segment is similar to other RNase III-sensitive sites found in E. coli and phage RNAs.

Integration host factor stimulates the phage lambda pL promoter.

Escherichia coli integration host factor (IHF) is a small dimeric protein that binds to a specific DNA consensus sequence and produces DNA bending. Transcription from the bacteriophage lambda pL promoter is stimulated three- to fourfold by IHF both in vivo and in vitro. IHF binds with high-affinity to two tandem sites located just upstream from the pL promoter and enhances the formation of RNA polymerase-promoter closed complexes. The rate of isomerization to open complex is not influenced by IHF. IHF may stimulate recognition of pL by one or more of several mechanisms: (1) by bending DNA; (2) by making protein-protein contacts with RNA polymerase; or (3) by occluding a competing promoter upstream from pL.

Genetic and biochemical analysis of the integration host factor of Escherichia coli.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal alpha-helix (alpha-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in beta-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation.

The bacteriophage lambda cIII gene product has a regulatory function in the lysis-lysogeny decision following infection. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding. By varying the temperature or Mg2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.

Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the alpha-subunit of RNA polymerase.

Escherichia coli integration host factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the alpha subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

Crystallization of recombinant chitinase from the cloned chiA gene of Serratia marcescens.

The chiA gene encoding for the chitinase enzyme from Serratia marcescens was efficiently overexpressed under the pL promoter and the enzyme was secreted into the growth medium. The chitinase was purified to homogeneity using affinity chromatography on a Phenyl-Sepharose column and the protein was successfully crystallized. The crystals are presently in the form of small needles in space group C222(1) and have unit cell dimensions a = 204(+/- 0.5) A, b = 134(+/- 0.5) A, c = 60(+/- 0.5) A. The crystals diffract X-rays to about 3 A resolution and are suitable for three-dimensional structural analysis.

Crystallization of recombinant chitobiase from Serratia marcescens.

We are currently investigating the biochemical and structural properties of both chitin degrading enzymes chitinase and chitobiase from Serratia marcescens. Previously we have reported the first crystallization and characterization of chitinase crystals (Vorgias et al., 1992). In this communication we present the first crystallization of chitobiase. The protein was synthesized in Escherichia coli and purified to homogeneity using cation exchange chromatography and fast protein liquid chromatography. The crystals have the shape of small prisms and the space group is P2(1) with beta = 101.0 degrees and unit cell dimensions a = 63.2 A, b = 133.2 A, c = 55.1 A. They diffract X-rays to about 2.5 A resolution and are suitable for three-dimensional structural analysis.

Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda.

An UP element defines a supplementary promoter element located upstream of the -35 region that stimulates transcription by interacting with the C-terminal domain of the RNA polymerase alpha subunit (alpha CTD). The alpha CTD also responds to various transcription activators, including the integration host factor protein, IHF, in the stimulation of the bacteriophage lambda PL promoter. PL consists of the tandem PL1-PL2 promoters where PL1 is stimulated and PL2 is repressed by IHF. We identified a functional UP element that binds the alpha subunit of RNA polymerase and is located in the region from -36 to -60 relative to the PL2 start site. PL2 expression requires the presence of the UP element and requires an intact alpha CTD. The UP element is nested within the DNA region protected by IHF against DNase I digestion. We used mutational analysis to identify the IHF recognition sequence which was found to be located downstream of the UP element, overlapping the -35 region of PL2. The possible function of the complex structure of the PL promoter is discussed.

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli.

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).

Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda.

The bacteriophage lambda cIII gene product is an early regulatory protein that participates in the lysis-lysogeny decision of the phage following infection. We have previously shown that the translation of the cIII gene is determined by two unique factors: (1) efficient expression is dependent upon the presence of RNaseIII in the cell; (2) alternative mRNA structures of the cIII coding region determine the rate of its translation initiation. In this study we demonstrate the presence of the alternative mRNA structures in vivo. The presence of minor RNaseIII cleavage sites within this region indicate that RNaseIII can differentiate between the two alternative structures. We localize by a deletion analysis the RNaseIII responsive element to the cIII coding region, and suggest that regulation of cIII translation by RNaseIII is achieved through binding to the alternative structures region of the mRNA.

Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540.

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.