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The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative. After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp. The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing. Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs). The BamHI J fragment was already known to encode IAP and OPDV-E6. Three other ORFs encode products similar to known proteins, viz. an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus σ-1 proteins. The ORF with similarity to σ-1 is also similar to baculovirus p10 proteins. In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

Asellus aquaticus is an isopod crustacean whose chromosomes cannot be differentiated by G- or R-banding techniques. In this work, we have obtained a longitudinal differentiation of these chromosomes by in situ nick translation using restriction enzymes (HaeIII, DraI and BamHI) and DNase I digestions. The four nucleases, with different efficiencies, have produced similar labelling patterns. Staining with DAPI, Giemsa and chromomycin A3 reveals that the DNA of the nick-translated regions is generally more resistant to extraction from the chromosome. The results obtained on the heteromorphic sex chromosome pair observed in about a quarter of the males of a natural population allow several hypotheses to be advanced on the nature and origin of chromosome dimorphism.

We previously reported the isolation of alphoid satellite clones from a human genomic library using a DNA immunoprecipitation with centromere protein B (CENP-B). Here, we have characterized the distribution of CENP-B-binding sites on the 3-kb BamHI repeats of the cos2 clone. Using in situ hybridization, this alphoid satellite was located primarily at the centromeric region of chromosome 6. The functional binding sites were mapped by precipitating the restriction fragments with recombinant CENP-B in vitro. One repeat (2B3-11) consisted of 19 copies of alphoid monomer, eight of which possessed the binding sites, while another (2B3-9) consisted of 18 copies of the monomer, seven of which possessed the binding sites. The distribution of the sites was well conserved between them, except for the terminus. A similar analysis with the remaining 6-kb region suggested the presence of a continuous 1-kb region with regular spacing of EcoRI sites and the CENP-B-binding sites. When the nucleotide sequence of 2B3-11 was compared with that of another chromosome 6-specific alphoid repeat (p308) that had been described previously, this 1-kb region was highly conserved between them. The distribution of the CENP-B binding sites and the order of alphoid monomers might define the folding of alphoid repeats in the centromeric region.

Restriction endonuclease cleavage patterns of mitochondrial DNA(mtDNA) of three local types of Yunnan native water buffalo were analyzed using 18 enzymes which recognize six nucleotides. Among the 12 animals analyzed, 3 of 18 enzymes, BamHI, EcoRI, and ScaI, revealed polymorphisms. Three mtDNA types were identified. The results indicate that a relatively low level of mtDNA variation exists in Yunnan domestic water buffaloes. The origin of Chinese buffalo derived from Yunnan province of China is discussed.

Here, a double-strand (ds) DNA microarray-based fluorescence assay has been deployed for studying endonuclease functionality and inhibition. The dsDNA microarrays are fabricated by hybridization of the Cy5-labeled oligonucleotides with the immobilized complementary oligonucleotide probes on the glass slide. The microarray displays significant fluorescence decrease in response to endonuclease, which is able to cleave the oligonucleotide moiety of dsDNA. Two endonucleases, EcoRI and BamHI, and four potential inhibitors, doxorubicin hydrochloride (DOX), 5-fluorouracil (5-FU), ethidium bromide (EB) and actinomycin D (ACTD), were selected to address the feasibility of this assay. Enzyme activities of the endonucleases are detected with high specificity down to the limits of 1.1 U mL(-1) for EcoRI and 2.0 U mL(-1) for BamHI, respectively. In addition, BamHI and EcoRI inhibition by the inhibitors are also shown, demonstrating the potential for high-throughput screening for inhibitors.

Soon after EBNA1 was found to be required to replicate and maintain oriP plasmids, EBNA1 was shown to bind to specific sequences in the oriP FR and DS elements through its C-terminal region (Jones et al. 1989; Rawlins et al. 1985). A screen for EBV genomic fragments bound by EBNA1 not only identified the FR and DS elements but also a fragment referred to as BamHI-Q (Jones et al. 1989), which was later shown to contain a promoter (Qp) used for the expression of EBNA1 in the absence of other EBNAs (Jones et al. 1989; Nonkwelo et al. 1996; Sample et al. 1992). EBNA1 was shown to have the highest affinity for the FR region, followed by the DS then the BamHI-Q region (Jones et al. 1989). All of the EBNA1-bound EBV fragments contained multiple copies of an 18 bp palindromic sequence that was protected by EBNA1 (Ambinder et al. 1990; Rawlins et al. 1985). The multiple copies of this sequence in the EBV genome are not identical but contain some variations that account for the different affinities of EBNA1 for the FR, DS and BamHI-Q regions and for individual sites within these regions (Ambinder et al. 1990; Summers et al. 1996). The BamHI-Q region contains two EBNA1 recognition sites that decrease EBNA1 expression when bound by EBNA1, enabling EBNA1 to autoregulate its own expression (Ambinder et al. 1990; Jones et al. 1989; Yoshioka et al. 2008).

The ability to accurately detect biomolecules in single-cell amounts is an important goal of basic and translational research. The inherent capability of nucleic acids to self-assemble allows the spontaneous formation of highly dense, self-assembled monolayers (SAMs). These assemblages can provide the basis for development of nano-based devices with programmable, single-molecule detection capability. Double-stranded(ds) RNA is an important biomarker of viral infection and certain cancers. While dsRNA behavior in solution has been extensively characterized by diverse physicochemical approaches, the properties of highly dense assemblies of dsRNA are largely unknown, and may be qualitatively different from those in solution. High-density, 39 bp, chimeric dsRNA:dsDNA (ds-chimera) SAMs have been prepared on modified gold surfaces. using atomic force microscopy (AFM)-based nanomanipulation and analysis, and fluorescence microscopy, we demonstrate the preferential binding of ethidium to the ds-chimera SAM compared to the single-stranded form. As revealed by AFM detection of height change, the ds-chimera SAM is reactive towards the dsRNA-specific RNase III of Aquifex aeolicus (Aa-RNase III) and restriction endonuclease BamHI, each having a recognition site in the dsRNA and dsDNA segments, respectively. We also show that the reactivity of the BamHI cleavage site, which is proximal to the gold surface, can be controlled by SAM density, and that access to the BamHI site is dependent upon the prior action of RNase III at its site in the center of the ds-chimera. These results reveal novel properties of protein-nucleic acid interactions within a high-density array environment that are relevant to nanoscale detection methodologies.

The Vibrio cholerae EITor typing phage D10 was characterized. The adsorption kinetics of the phage on V. cholerae MAK757 strain were biphasic in nature. Intracellular growth was characterized by an eclipse period, latent period and burst size which were 20 min, 25 min and 80 particles per cell respectively. The phage yield was dependent on the concentration and time of addition of DNA synthesis inhibitors such as nalidixic acid and novobiocin, and RNA synthesis inhibitors such as rifampicin. The 32+/-0.2 kb linear double-stranded DNA molecule has unique termini. A restriction map of the phage DNA was constructed with the enzymes BamHI, HindIII and PstI.

In Epstein-Barr virus (EBV)-associated tumors that arise in immunocompetent individuals, the pattern of viral gene expression is very restricted compared with that of latently infected B cells in tissue culture. A hallmark of viral gene expression in these tumors is the exclusive expression of only one EBV-encoded nuclear antigen, EBNA1, which is driven from a promoter (Qp) that lies near the junction of the viral BamHI F and Q fragments. During induction of the lytic cycle, a viral promoter, Fp, which lies ca. 200 bp upstream of Qp, gives rise to transcripts which overlap with Qp-initiated EBNA1 gene transcripts. Distinguishing between latency-associated EBNA1 gene transcripts and those associated with the early phase of the viral lytic cycle is critical for correct identification of restricted viral latency. Here we describe a reverse transcriptase PCR protocol which employs a nested set of upstream primers from the BamHI Q region of the viral genome and readily distinguishes Fp-initiated transcripts from Qp-initiated transcripts. A single set of amplification conditions was used for the various PCR primer combinations, which allowed all reactions to be run simultaneously. An in vitro-generated transcript, diluted in RNA from an EBV-negative cell line, was used to demonstrate that the efficiencies of amplification with the different primer combinations were very similar. This protocol was used to demonstrate that EBNA1 gene transcription in two previously uncharacterized EBV-positive epithelial cell lines initiates from Qp. In addition, we assessed the site(s) of initiation of EBNA1 gene transcripts in cell lines exhibiting restricted viral latency. Contrary to the results of Nonkwelo et al. (J. Virol. 70:623-627, 1996), which indicated that EBNA1 gene transcription during restricted viral latency initiates at multiple sites downstream of Fp, we show here that nearly all EBNA1 transcripts start at the previously identified Qp transcription initiation site.

DNA sequence-reading bisquaternary ammonium heterocycles SN 6570, SN 6999, SN 6053, SN 6132, SN 6131, SN 18071 and the non-specific binders SN 6113, SN 5754, SN 6324, and SN 4094 influence the enzymatic activity of restriction endonucleases in different manners. A prerequisite for sequence-specific ligand interaction is a dAdT run of at least four base pairs. The sequence-specific binders inhibit the cleavage activity of restriction endonucleases EcoRI, SspI, and Dral with four and six dAdT base pairs in their restriction sites, while the activity of SalI and BamHI with less than four dAdT base pairs in their recognition motifs remains unaffected. On the contrary, the non-specific binding DNA ligands are incapable of suppressing the digestion for restriction nucleases under research. These results are in line with our footprint data. The inhibitory effect is independent of the number of cleavage sites in DNA and of whether the macromolecule exists in the ccc or lds conformation. Sequence specific binding of the ligand SN 6053 in close vicinity to the cleavage sites of restriction endonuclease Dral also interferes with enzyme inhibition.

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.

We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXI/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (t1-mRNA) by trans-splicing, the cells utilize a 5′ cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3′ splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5′ splice site. The T1-mRNA was not generated when the cryptic 5′ splice site was mutated.

An inducible marker system suitable for Agrobacterium tumefaciens was constructed to enable detection and enumeration of specific bacterial cells introduced into soil. A BamHI cassette carrying the catechol 2,3-dipxygenase (C230) gene, tdnC, fused to the nopaline-inducible Agrobacterium promoter Pi 2[noc] was constructed. This cassette was introduced into the broad host range vector pDSK5019 resulting in plasmid pTVNC2. Inducible C230 activity was observed in an A. tumefaciens strain carrying the plasmid pTVNC2 when nopaline was present. Colonies of bacteria tagged with the system could easily be identified by spraying agar plates containing nopaline with catechol, which is converted to the bright yellow compound 2-hydroxymuconic semialdehyde. The inducible tdnC cassette has also been introduced into the BamHI site of the transposon Tn5 carried by the pSUP1011 suicide vector which can be used as a delivery system for the stable introduction of the inducible marker into the chromosome of target cells.

We describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406-0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome. The fragment contains three nested genes with a common 3' end. The 5' ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped. Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5. ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene. The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor. ORF2 was identified as the PRV proteinase gene following expression in E. coli, using the product of ORF2.5 as the substrate protein.

A 16.5-kb BamHI fragment of the Cellvibrio mixtus chromosome was found to direct carboxymethylcellulase, xylanase, and avicel hydrolysis. Two closely linked genes were subcloned from this insert. The gene, cmcI, was cloned as a 2.7-kb fragment and expressed in Escherichia coli. It encoded an enzyme of approximately 74 kDa which degraded carboxymethylcellulose and xylan but did not attack the microcrystalline cellulose substrate avicel. A second cellulase capable of degrading avicel, encoded by exoI, was found 5.5 kb downstream of cmcI. Two translation products of 53.7 kDa and 51.5 kDa were produced in E. coli strains expressing exoI. Northern analysis of total mRNA of C. mixtus grown on avicel, with a probe generated from cmcI, showed that cmcI and exoI were not cotranscribed in an operon.

The genomes of pseudorabies virus (PrV) and of herpes simplex virus type 1 (HSV1) are colinear, excepting an inversion in the unique long region, of which one extremity resides within the BamHI fragment 9. This fragment (4088 bp) encodes the counterparts of HSV1 UL24, UL25, UL26 and UL26.5 that are transcribed into four 3'-coterminal mRNAs. Multiple alignments of UL24, UL25 and UL26 protein homologs from alpha-, beta- and gamma-herpesviruses were performed. The PrV UL24 protein is shorter than its counterparts, missing the non-conserved COOH-terminal region. The region which is common to all viruses contains a basic NH2-terminus and a hydrophobic COOH-end, suggesting that UL24 may function as a matrix protein. The UL25 proteins are well conserved, particularly among the alpha-herpesviruses. All the domains involved in the proteolytic activity of theUL26 protein are highly conserved, as well as the two cleavage sites. Thus, its function and processing may be similar in PrV as in other herpesviruses. Due to the fact that in PrV the UL26 and UL44 genes are adjacent and their ends are conserved, the right border of the inversion must lie within their intergenic region.

Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping. Several enzyme combinations, including the best ones proposed in literature, were utilized and the highest discrimination was reached by individual digestion with PvuII, HindII, and EcoRI or BamHI. Clinical isolates from outbreaks were clonally related as identified by this molecular approach. Restriction rDNA profiles were composed of strong and weak bands. Using 6 micrograms DNA we were able to demonstrate that PvuII, HindIII, and BamHI weak bands were reproducible. These weak bands should be considered not only to accomplish the highest discrimination but also to correctly assign isolate clonality. Conversely, we found that EcoRI weak bands were not reproducible and, therefore, are not recommended for ribotype analysis. Finally, profiles differing in one single band actually represented isolates of different genotype, as confirmed by further analysis using other molecular methods. In this report on P. Aeruginosa ribotyping of clinical isolates, criteria for band pattern interpretation are established.

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage phi 20 isolated from B. anthracis Sterne 34F2 (pXO1+ pXO2-) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The phi 20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.

A restriction fragment library representing 89.3% of the genome of Trichoplusia ni granulosis virus (TnGV) was constructed. The library consisted of 13 of the 16 BamHI fragments, 18 of the 22 EcoRI fragments, and 6 of the 27 PstI fragments. By restriction endonuclease and Southern blot analysis of cloned or genomic viral DNA fragments, a complete physical map of TnGV was constructed for BamHI, EcoRI, PstI and XhoI. Three interspersed homologous regions (ihs1-ihs3) were identified from hybridization experiments and sequenced. Each TnGV ihs has an approximate size of 400 bp and shows homology to the other two. The orientation of ihs2 is inverted relative to ihs1 and ihs3. TnGV ihs regions do not have repetitive motifs or palindromic sequences, in contrast to homologous regions (hrs) of nuclear polyhedrosis viruses (NPVs). The genomic locations of TnGV ihs1-ihs3, represented in percentage map units, were very similar to those of ihs sequences previously reported in Bombyx mori NPV, suggesting that the ihs may be a novel type of cis-acting element common among baculoviruses. Additionally, an inverted repeat sequence, having overlapping, multiple inverted repeats of 400 bp, was identified to the left of ihs3 on the linearized genome map of TnGV.

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

Summary Four different clones, PG2, PG3, PG5 and PG6, were isolated by screening a cosmid library of an inbred homozygous SLA typed pig with a human MHC class I cDNA probe (Hertner 1991). These clones were mapped with four different restriction endonucleases (EcoRI, BamHI, HindIII and KpnI). After hybridization, nine SLA class I genes were identified. By transfection of the four clones into Ltk- cells, the expression of the SLA class I genes was measured cytometrically using an SLA specific antiserum. To differentiate between classical and-non-classical genes, the exons 2 and 3 of the 9 genes were sequenced. After determination of the G/C content at the third codon position and comparison with sequences of published SLA class I genes, three of them could be identified as classical genes. A specific primer was constructed for one of these classical class I genes and used to amplify and differentiate the exon 2 of 200 animals from four different German pig breeds. Zusammenfassung Molekulargenetische Charakterisierung der MHC Klasse I Region beim Schwein Vier unterschiedliche Klone PG2, PG3, PG5 und PG6 wurden durch Screening der Cosmidbank eines in seinen SLA-Haplotypen homozygoten Hausschweines mit einer humanen MHC-Klasse I-cDNA-Sonde isoliert (Hertner 1991). Eine Restriktionskarte fur diese Klone wurde mit vier verschiedenen Endonukleasen (EcoRI, BamHI, HindIII und KpnI) erstellt. Durch Hybridizierungsversuche liesen sich neun SLA-Klasse I-Gene identifizieren. Nach der Transfektion der vier Klone in Ltk- Zellen wurde die Expression der SLA-Klasse I-Gene mit Hilfe eines spezifischen Antiserums cytometrisch gemessen. Um die neun Klasse I-Gene den ‘klassischen’ oder ‘nicht klassischen’ Genen zuzuordnen, wurden die hoch polymorphen Exon 2- und 3-Regionen sequenziert. Nach Bestimmung des G/C-Gehaltes an der dritten Codonposition der sequenzierten Regionen und des Vergleichs mit den publizierten Sequenzen der SLA-Klasse I-Gene wurden drei Gene als ‘klassisch’ zugeordnet. fur ein ‘klassisches’ Klasse I-Gen wurde ein spezifischer Primer konstruiert, mit dem die Exon 2-Region von 200 Tiere aus vier verschiedenen Schweinerassen amplifiziert wurden.

Ribosomal DNA polymorphism was studied in order to demonstrate intra- and interspecies differentiation of 42 Bifidobacterium strains. DNA from these strains was digested with the endonucleases BamHI, EcoRV, HindIII and PvuII and then analysed by Southern blotting. Ribosomal patterns using a part of an rRNA 23S gene as a probe clearly differentiated the majority of species from each other. Only B. indicum ATCC 25912T and B. infantis ATCC 15697T displayed identical ribosomal patterns, even though they are classified into two different species. Moreover, ribotypes were able to distinguish between strains belonging to the same species. Furthermore, these strains generally showed common bands, except for B. infantis strains and two strains of B. animalis., L'ADN ribosomique de 42 souches du genre Bifidobacterium a été étudié afin de démontrer leur variabilité intra- et interspécifique. L'ADN de ces souches a été digéré par les endonucleases BamHI, EcoRV, HindIII and PvuII puis analysé par Southern blot. Les profils ribosomiques obtenus par l'utilisation d'une sonde correspondant à une fraction du gène ribosomique 23S différentie clairement la majorité des espèces. Seules les souches B. indicum ATCC 25912T et B. infantis ATCC 15697T présentent le même profil abien qu'elles aient été classées dans des espèces différentes. Les ribotypes obtenus permettent également de distinguer des souches de la même espèce. Les profils de ces dernières présentent le plus généralement des bandes communes, exceptées celles de l'espèce B. infantis et les deux souches de B. animalis.

Wheat varieties, differing in chapati characteristics, showed marked restriction fragment length polymorphism when probed for gene encoding α-gliadin. EcoRI digested DNA from variety K-68 showed five hybridizing bands whereas Sonalika showed only three bands. BamHI and HindIII digested DNA from variety C-306 showed lesser number of hybridizing bands than Sonalika, while Pst I digested K-68 DNA showed six hybridizing bands. By screening of sub-genomic library of C-306, 10 clones encoding gliadin were isolated. Partial sequencing of a clone pBJ-1 (∼ 1.0 kb) showed polyglutamine coding region and an internal stop condon at 224 bp.

Southern hybridization analysis of the IncC plasmid pDGO100 showed that, in addition to the well-characterized integron In7, there is a second integron which is located on a 3.6-kb BamHI fragment. This integron also possesses the qacE delta l and sulI genes typically found as part of the 3'-conserved segment of integrons. The 3.6-kb BamHI fragment, when cloned into pUC19 to form pDGO301, conferred resistance to trimethoprim as well as sulfamethoxazole. The DNA sequence of the trimethoprim resistance gene in pDGO301 was determined and it was shown that the gene was precisely inserted as a cassette in an integron with 100% identity to the trimethoprim resistance gene dfrA7.

The etiology of congenital generalized lipodystrophy (CGL) is unknown, but one of the clinical features is an accelerated rate of growth. In order to determine whether this abnormality is related to an abnormality of the GH or the IGF-I gene, we studied the DNA of lymphocytes of a girl with CGL. The IGF-I gene autoradiographic patterns of this patient were found not to differ from those of normal subjects, but after BamHI digestion, this patient's GH gene cluster had an aberrant 5.7 kb fragment in addition to the constant 8.3, 6.7, 5.3, 3.8, 2.9 kg fragments. The new 5.7 kb fragment was not detected in any of 60 unrelated Japanese individuals. In conclusion, the CGL patient carries an abnormal GH gene cluster, but further studies are needed to clarify whether this abnormal fragment is specific to CGL or just a rare polymorphic fragment.

Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO(+), ddY/DAO(-), ICR, NC, NZB and NZW) for the examination of the difference in the structure of theD-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingD-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO(+) and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similarD-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.

The dnaK operon of Streptomyces coelicolor A3(2) was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two of the most conserved regions in 30 different DnaK proteins (HSP70). The isolated insert-a BamHI 5.6-kb fragment-was sequenced and shown to contain three open-reading frames organized in an operon and coding for proteins analogous to DnaK, GrpE and DnaJ, successively.

Klebsiella pneumoniae 2207, from Durban, was resistant to cefoxitin and beta-lactamase inhibitor combinations as well as oxyimino-aminothiazolyl cephalosporins. Beta-lactamases with isoelectric points of 5.4, 5.6, 7.6, 8.2 and 8.4 were found. DNA hybridisation identified two BamHI and three HindIII fragments carrying blaTEM, and two SalI fragments carrying blaSHV. At least two genes encoded TEM-1 enzyme; one blaSHV copy encoded SHV-5 but the other determined SHV-23, a novel SHV-5 variant with conservative amino-acid substitutions far from the catalytic site. The pI 8.4 activity was an AmpC-type enzyme. Determinants of the pI 5.6 and 7.6 activities were not identified.

A pair of proper primers were designed and synthesized for Polymerase Chain Reaction (PCR) of anti-bacterial gene aiiA which encodes protein aiiA from marine bacterial genome, according to the gene sequences of aiiA. The plasmid pMD18-ZD02aiiA was constructed after PCR testing of aiiA gene. The gene sequence of aiiA was amplified with proper primers (with active locations by enzymes BamHIand EcoRI) from molding board of pMD18-ZD02aiiA and linked into expression vector pET-17b after it was hydrolyzed by enzymes of BamHI and EcoRI. And then plasmids pET-ZD02aiiA was constructed. The results of hydrolyzation of enzymes, PCR amplification and sequencing demonstrated that the target gene fragments were inserted into vector pET-17b correctly, the sequences of gene aiiA and frame of reading codes were right. Thus, it may provide base for the recombinant express and induced express of anti-bacterial gene from marine bacterium.

Seven wildPetunia species including 2n = 18 species (P. parviflora Jussieu,P. linearis Hook.) and those with 2n = 14 (P. parodii Steene,P. axillaris Lam.,P. integrifolia Hook.,P. inflata R.E. Fries,P. violacea Lindl.) and tenPetunia hybrida horticultural lines were compared for polymorphisms in rDNA genes using the four restriction enzymesEcoRI,BamHI,HindIII andXhoI. All the unit types found in the lines pre-existed in the wild forms. There are two different sizes of either 11.45 or 11.6 kb./The 2n = 18 species are closely related to the 2n = 14 species, thus making thePetunia genus homogeneous. Moreover, it is likely thatP. hybrida lines originated in several kinds of crosses between these species. We constructed a dendrogram for all the 15 rDNA unit types found. Two main branches of the tree result from the presence or the absence ofHindIII sites. The main branch is divided according to variability at theEcoRI andBamHI sites. Taking into account the existence of several loci which carry one unit type only, we consider whether or not exchanges might occur between loci. Lines carrying two unit types and lines carrying three unit types support such a hypothesis.XhoI andBamHI fragments enable us to distinguish two types of 5S DNA corresponding to 2n = 18 and 2n = 14 species, respectively.P. hybrida lines and each 2n = 14 wild species carry one of the types only, that corresponds to one 5S DNA locus. The most parsimonious phylogenetic trees whatever the species chosen as the outgroup, do not fit with our knowledge ofPetunia and with taxonomy. This is likely because only few loci formed the basis of these phylogenetic constructions.

We have isolated and sequenced two cDNAs of sizes 2674 and 677 bp from a cDNA library derived from MKT-1, a lymphoblastoid cell line transformed and latently infected with Marek's disease virus (MDV) using probes corresponding to the right-hand end of the BamHI-I2 fragment of the MDV genome. The larger cDNA clone represents an abundant transcript, which extends from the right-hand end of BamHI-I2 to the adjacent BamHI-Q2 and BamHI-L fragments of the MDV genome and contains the Meq (MDV Eco-Q) open reading frame. The smaller cDNA clone represents a spliced transcript containing the putative DNA binding domain of Meq as well as sequences in the BamHI-L region. We prepared a polyclonal antibody against part of the protein sequence of Meq and detected a 44-kDa protein in MKT-1 cells and in cells lytically infected with MDV. In addition, riboprobes corresponding to sequences specific to each cDNA as well as shared sequences between cDNAs detected a number of transcripts in cells either lytically or latently infected with MDV. Our results indicate that the Meq transcriptional unit extends to the BamHI-L fragment and that the transcripts mapping to the right-hand end of the BamHI-I2 and adjacent BamHI-Q2 and BamHI-L fragments are not preferentially expressed during latency.

This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.

A targeted mapping strategy using representational difference analysis (RDA) was employed to isolate new restriction fragment length polymorphism probes for the long arm of chromosome 6 in rye (6RL), which carries a gene for resistance to Hessian fly larvae. Fragments from the 6RL arm were specifically isolated using a 'Chinese Spring' (CS) wheat – rye ditelosomic addition line (CSDT6RL) as tester, and CS and (or) CS4R as the driver for the genomic subtraction. Three RDA experiments were performed using BamHI amplicons, two of which were successful in producing low-copy clones. All low-copy clones were confirmed to have originated from 6RL, indicating substantial enrichment for target sequences. Two mapping populations, both of which are derived from a cross between two similar wheat–rye translocation lines, were used to map five RDA probes as well as five wheat probes. One of the populations was prescreened for recombinants by C-banding analysis. Fifteen loci, including seven new RDA markers, were placed on a map of the distal half of 6RL. The Hessian fly resistance gene was localized by mapping and C-banding analysis to approximately the terminal 1% of the arm. The utility of RDA as a method of targeted mapping in cereals and prospects for map-based cloning of the resistance gene are discussed.Key words: Secale cereale L., Hessian fly resistance; RDA, RFLP, high density mapping.

Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3′ to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MfII, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Epstein-Barr virus (EBV) gene expression in nasopharyngeal carcinoma (NPC) includes abundant rightward transcription of the BamHI A fragment, consisting of mRNAs ranging in size from approximately 4.0 to 8.0 kb. These transcripts include several distinctly spliced forms which are 3'-end coterminal and contain the BamHI A rightward frame 0 (BARF0) open reading frame (ORF) in the final exon. BamHI A transcription is detected at a lower level of expression in EBV-infected lymphoid cells. In this study, cDNA cloning, reverse transcription-based PCR, and Northern (RNA) blotting were used to further define the structures of the BamHI A transcripts and to characterize their expression in different EBV-infected tissues. Three BamHI A cDNAs isolated from a passaged NPC represent previously unidentified mRNAs that contain BARF0 and additional ORFs encoded by multiple exons, including one which extends the size of the BARF0 ORF from 174 to 279 codons. The distinct exons were detected in multiple, differently sized mRNAs, indicating that these transcripts have complex patterns of alternate splicing. In support of this finding, 5'-end analysis confirmed the presence of a previously reported start site and also identified a subset of transcripts of 4.8 kb and larger that initiate further 5' to this site. In addition, 3'-end analysis identified heterogeneous 3'-end processing in all of the BamHI A mRNAs, resulting in transcripts that either contain the entire BARF0 ORF or are cleaved and polyadenylated 5' of the stop codon. Finally, the expression of multiple, distinctly spliced BamHI A transcripts was consistently detected in a wide range of EBV-infected samples, including NPC, Burkitt's lymphoma, and parotid carcinoma biopsy samples, and in type I and type III Burkitt's lymphoma lines and type III lymphoblastoid cell lines. This complex pattern of start site selection, alternate splicing, and heterogeneous 3'-end processing is likely to regulate the expression in vivo of the ORFs encoded by the EBV BamHI A transcripts.

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.

Supercoiled pUC19 DNA, photoreacted with psoralen derivatives (xanthotoxin (8-methoxypsoralen, 8-MOP), 4,5′,8-trimethylpsoralen (TMP) and angelicin), influences the enzymatic activity of restriction enzymes in a different manner, although all the enzymes employed contain, within their recognition sites, suitable nucleic acid bases for photoproduct formation. The activity of the enzymes is strongly influenced by the photomodification of thymine residues within their recognition sites. 5′-TpA sequences favour intercalation as an essential prerequisite of the photoreaction, while 5′ApT sequences do not. This, in turn, influences photoproduct formation and the inhibition of the action of the restriction enzymes KpnI, SspI, DraI and RsaI, but not EcoRI and BamHI. The inhibitory effect is independent of the number of cleavage sites and also of whether monoaddition products or crosslinks are formed. Psoralen intercalation alone does not affect the activity of the restriction enzymes used.

Mitochondrial DNAs (mtDNAs) were purified by CsCl/bisbenzimide density-gradient ultracentrifugation from 21 strains of seven Fusarium species that cause fusarium head blight and mycotoxin contamination in wheat and other cereals. A partial PstI clone bank, from which one of twelve PstI fragments (14.7 kb) is missing, was constructed using mtDNA from strain KU-1615 of F. graminearum. Molecular sizes of mtDNAs of single representative strains from the seven species were determined after single-, double- and triple-digestion by four or five restriction enzymes (BamHI, MluI, PstI, PvuII and XhoI), while those of others were after single-digestion by BamHI and/or PstI. MtDNA size varied from the smallest 49 kb in one strain of F. avenaceum to the largest 116 kb in one strain of F. culmorum. Restriction fragment length polymorphism (RFLP) analysis revealed a large interspecific variation, thus all the species were identified by their restriction fragment patterns and assigned to individual clusters except for F. tricinctum in that a strain studied showed identical patterns to one of two strains of F. sporotrichioides. Considerable intraspecific variation including size variation was also detected. These results indicated a high incidence of insertions/deletions both between and within species. On the basis of results obtained by the cluster analysis, some aspects of taxonomy in these Fusarium species were discussed.

Optical Mapping, a single DNA molecule genome analysis platform, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule1. Understanding how the cyanine dimer fluorochromes affect enzyme activity can lead to modifications to the Optical Mapping System. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously (1). However, TOTO-1 is apart of a family of cyanine dimer fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. Therefore, lambda DNA was stained with one of the cyanine dimer fluorochromes and then digested with a restriction enzyme (BamHI, SmaI, ScaI-HF, HindIII, SgrAI, EcoRI, or PmlI). The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine if there was complete digestion, incomplete digestion, or inhibited digestion. Some enzymes were inhibited by the YOYO-1, TOTO-1, and POPO-1 fluorochrome.1. Meng, X.; Cai, W.; Schwartz, D.C.; 1996, Journal of Bimolecular Structure and Dynamics, 13, 945.

Protein–protein interactions are essential for signal transduction in cells. Bimolecular fluorescence complementation (BiFC) is a novel technology that utilises green fluorescent proteins to visualize protein–protein interactions and subcellular protein localisation. BiFC based on pSATN vectors are a good system for high-level expression of fused protein. A series of pCAMBIA vectors were most widely used in plant transgene and transient expression. To provide multiple options in the study of protein interactions that utilise BiFC, we reconstructed a new pair of BiFC vectors, pCAMBIA1301-nEYFP and pCAMBIA1301-cEYFP. These vectors were generated by eliminating restriction enzyme cutting sites (BanII, SacI, KpnI, SmaI, BamHI, SalI, PstI and SbfI) at the multiple cloning sites (MCSs) of pCAMBIA1301 (p1301), and introducing cEYFP/nEYFP cassettes containing MCSs generated from pSATN medium. Fluorescence can be imaged when AtCBL1 and AtCIPK23 are co-injected, but imaging cannot be done when co-injecting AtCBL1 and AtCIPK23-NAF-deleted (AtCIPK23m), suggesting that the proposed modified vector system is effective for the study of protein interactions.Keywords: Protein–protein, bimolecular fluorescence complementation (BiFC), vector reconstruction

In this study an impedimetric DNA sensor has been used for the detection of protein-DNA interactions. The sensor is based on single-stranded DNA bound to gold chip electrodes. In the presence of ferri/ferrocyanide impedance measurements show an increase in charge transfer resistance Rct after immobilization and hybridization of ssDNA to the sensor surface. This allows hybridization detection in the low nanomolar concentration range. This electrode system is used to follow the sequence specific reaction of the restriction endonuclease BamHI on the surface electrochemically. Furthermore, the sequence specific DNA-binding of the transcription factor NF-κB p50 is found to cause a decrease in Rct resistance in a concentration dependent way.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).

Fluorescently labeled oligonucleotides and DNA fragments have promise in nucleic acid research with applications that include DNA hybridization, automated DNA sequencing, fluorescence anisotropy, and resonance energy transfer studies. Past concerns with fluorescent-labeled DNA arose from interactions between fluorophores and DNA that result in quenched fluorescence. This quenching phenomenon is most problematic in fluorescence resonance energy transfer studies because quenching of the donor fluorescence could result from either resonance energy transfer or nontransfer effects. In the present study, relief of nontransfer quenching of a 14-mer fluorescein 5-isothiocyanate (FITC)-labeled oligonucleotide containing the BamHI restriction site was characterized with both steady-state and time-resolved fluorescence techniques. The FITC-labeled single strand was best fit by a triexponential decay with lifetimes of 0.5, 2.7, and 4.2 ns. The 4.2-ns component was found to contribute more than 80% of the total steady-state intensity. Upon annealing with an unmodified complementary strand, the contribution from the 4.2-ns component was significantly decreased, resulting in twofold quenching of total fluorescence. We reasoned that this quenching phenomenon should be a reversible process and could be employed to study strand separation processes in molecular biology. Hence, cleavage of the fluorescently labeled substrate was examined using DNAase I and BamHI restriction endonuclease. Our results show that the quenched fluorescence is totally recovered upon cleavage (compared to that of the single strand). The extent of cleavage measured by fluorescence was confirmed by nondenaturing polyacrylamide gel electrophoresis analysis. We believe this fluorescence "dequenching" technique may be used to quantify the kinetics of other DNA strand separation and cleavage processes in molecular biology.

We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.

A set of vectors has been constructed to facilitate the fusion of heterologous sequences to the C terminus of the maltose-binding protein (MBP) of Escherichia coli. The plasmids carry a cloning region comprising two blunt cloning sites, a BamHI site and multiple stop codons, and this has been placed in each reading frame so that translational fusions to MBP can be generated and manipulated with ease. To demonstrate the utility of this system, recombinant proteins have been engineered in which staphylococcal enterotoxin A has been fused to MBP.

TYPE II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified1,2. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.

RFLPs of mtDNA for HpaI, BamHI, HaeII, MspI, AvaII and HincII were analysed in a sample of 100 from collected in Tirana (Albania). Eighteen mtDNA types were identified, four of which are new: two because of new morphs for Hpa/HincII (HpaI-9Tir/HincII-17Tir) and AvaII (AvaII-35Tir), and two because of new associations between morphs which were already known. Albanian data were compared with corresponding ones from Calabrians (Southern Italians) and Arbreshes (people of Albanian ancestry living in Calabria). Even though the examined mtDNA markers do not reveal important differences among the three groups, the analysis of both morphs and types shows a trend which places Arbreshes closer to Calabrians than to Albanians.