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Biomedical subjects

H Malke

Publications and source records attributed to H Malke.

At least 55 records · Page 3Linked to original sources
PubMed1988

Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative procaryotes.

A specific 1,596 bp HincII fragment ('skc) from the chromosome of Streptococcus equisimilis contains an active streptokinase (SK) gene (skc) lacking, in addition to the expression signals, codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator. Using this fragment as an indicator gene, we constructed two types of vectors which in appropriate hosts resulted in the synthesis of SK fusion proteins after insertional activation of 'skc. The first type are open reading frame (ORF) vectors in which 'skc was inserted into pUC18 out of frame with respect to lacZ', thus conferring an SK-negative phenotype. Any DNA fragments representing ORFs inserted between the lacZ' expression signals and 'skc such that the skc reading frame was restored resulted in the production of tripartite proteins which exhibited SK activity. The second type of vector, which functioned in both gram-positive and gram-negative bacteria, used the streptococcal speA expression and secretion signals in front of the ORF to activate 'skc insertionally. Using a large fragment from the chymosin gene as the target sequence, the usefulness of these vectors for studying foreign gene expression in streptococci as well as Escherichia coli was demonstrated.

Amino Acid Sequence↗
PubMed1987

Improved treatment results in childhood acute nonlymphoblastic leukemia with the BFM-AML protocol 78 in a multicenter study in the GDR.

Eighty-seven children with acute nonlymphoblastic leukemia were treated with the AML protocol BFM 78 between June 1979 and February 1986 in a multicenter study in the GDR. Seventeen children (20%) died from early complications, eight did not respond to therapy. Fifty-eight patients (70%) achieved a complete remission. Twenty-three patients relapsed. The life table analysis revealed after 5 years a probability for event-free survival of 36% (SD = 6%) and an event-free interval of 51% (SD = 8%). Six patients were transplanted in first remission. Two of them died; one (M 1) on day + 19 from encephalopathy and one (M 4) on day + 60 from acute GVHD. The overall results are in good correlation with the original BFM study, but there are differences in the subtypes. Results are superior to other AML protocols in our group.

Actuarial Analysis↗
PubMed1987

Results of acute lymphoblastic leukemia therapy in childhood with a modified BFM protocol in a multicenter study in the German Democratic Republic.

Between 1 September 1981 and 31 December 1985, 382 previously untreated children with ALL were entered into study VII/81, a multicentric and randomized study with a modified BFM protocol. Patients were divided into three risk groups according to the initial lymphoblast count and liver and spleen enlargement: standard- (SR), medium- (MR), and high-risk (HR) groups. Of all patients, 94% attained complete remission. The actuarial probability of event-free survival is 0.62 +/- 0.04 (SR group, 0.66 +/- 0.06; HR group, 0.29 +/- 0.12). Sixty-one patients relapsed, 10 had isolated CNS relapses, and 11 CNS relapses were combined with bone marrow relapses. Concerning the duration of maintenance therapy, patients were randomized into two groups of 18 and 24 months respectively. Up to now, there has been a slight advantage for the 18-month group. Two different methods of CNS preventive therapy for SR patients (irradiation plus intrathecal methotrexate and intermediate-dose methotrexate (IDMTX) plus intrathecal methotrexate) were used and revealed a higher rate of CNS relapses but a lower rate of bone marrow relapses in the intermediate-dose MTX group.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues.

The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W., & Ferretti, J.J. (1984) MGG, Mol. Gen. Genet. 196, 360-365] while the molecular weight of the native streptokinase is 47 000. The structural and activity differences of the cloned streptokinase (cSK) as expressed by S. sanguis and the native streptokinase (nSK) were investigated. From a partially purified cSK, two active fractions were obtained by reversed-phase HPLC. The minor fraction cSKL was nearly as active as SK in plasminogen activation. The major fraction cSKs had only about one-fourth of the specific activity. The structures of cSKL and cSKs were studied and compared to the known amino acid sequence of SK [Jackson, K. W., & Tang, J. (1982) Biochemistry 21, 6620-6625]. From the NH2- and COOH-terminal sequences and amino acid composition of the cyanogen bromide (CNBr) fragments, it could be deduced that cSKL and cSKs are without 31 and 32 residues, respectively, from the COOH-terminal end of SK. Since the cloned gene contained the full SK structure, the missing structures must have been due to posttranslational proteolysis. An SK fragment similar in size to cSK was observed from a chymotryptic digest of SK.

Amino Acids↗
PubMed1986

Expression of the streptokinase gene from Streptococcus equisimilis in Bacillus subtilis.

The previously cloned and sequenced streptokinase gene (skc) from Streptococcus equisimilis H46A was inserted into plasmid vectors capable of replication in Bacillus subtilis. The skc gene was expressed by use of its own transcription and translation signals which appeared to meet the stringent requirements of B. subtilis for efficient foreign gene expression. The secreted streptokinase activity began to decline toward the end of the exponential growth phase suggesting that B. subtilis exoproteases hydrolyzed and inactivated the foreign protein.

Bacillus subtilis↗
PubMed1986

Codon usage in streptococci.

Codon usage was analysed for 14 streptococcal genes or significant open reading frames and found to be different from that in Escherichia coli and Bacillus subtilis. In particular, the preferred use of WWT codons over WWC was inconsistent with the rule of optimal codon-anticodon interaction energy. On the other hand, for SSTC codons, adherence to this rule was better in streptococci than in E. coli. A preliminary codon bias table generated with the Pustell computer program for the analysed streptococcal genes may prove useful for the detection of protein coding regions in newly sequenced DNAs from both streptococci and staphylococci.

Bacillus subtilis↗
PubMed1986

The streptokinase gene.

The subject of this paper is the molecular cloning, nucleotide sequencing, and expression in heterologous hosts of the streptokinase gene (skc) from the group C streptococcal strain H46A. The skc gene shows no extended regions homologous to the staphylokinase gene.

Base Sequence↗
PubMed1985

Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184.

The streptococcal erythromycin resistance (Emr) plasmid pSM7 (6.4 kb) and the E. coli vector pACYC184 (4.0 kb) were fused at their single EcoRI sites to form the bifunctional chimeric plasmid pSM7184 (10.4 kb) in which the Emr determinant was placed under control of the chloramphenicol acetyl transferase (cat) promoter of pACYC184. In the sense orientation (orientation I) of pSM7, the cat promoter directed expression of Emr in the E. coli host strains 294 and DB11 more efficiently than did the indigenous transcription signals of pSM7, which were functional in the opposite orientation II. In Streptococcus sanguis (Challis), the level of Emr was independent of the orientation of pSM7 in pACYC184, showing that the cat promoter was not recognized in the gram-positive host. The growth of E. coli (pSM7184I) in a defined medium containing glycerol as carbon source, or containing glucose plus extraneous cyclic 3'-5' adenosine monophosphate (cAMP) led to an Emr level which was 15-30 times higher than that of cultures grown on glucose. These results showed that under control of the cat promoter, Emr is subject to cAMP-mediated catabolite repression and provided conclusive evidence that the enhancement of Emr expression in E. coli carrying pSM7184I is controlled at the transcriptional level. Besides enabling us to determine the orientation of transcription of the Emr gene in pSM7 and related vectors, this work also made available new bifunctional cloning vehicles able to replicate in both E. coli and S. sanguis.

Acetyltransferases↗
PubMed1985

Nucleotide sequence of the streptokinase gene from Streptococcus equisimilis H46A.

The entire nucleotide sequence of a cloned 2568-bp PstI fragment from the genome of Streptococcus equisimilis H46A encoding the streptokinase gene (skc) has been determined. The longest open reading frame comprises 1320 bp which code for streptokinase. The protein is synthesized with a 26-amino acid residue N-terminal extension having properties characteristic of a signal peptide. Comparison of the deduced amino acid sequence with the available amino acid sequence of a commercial streptokinase reveals minor primary structure differences. The nucleotide sequencing of skc does not support the hypothesis that the gene has evolved by duplication and fusion, as suggested by internal twofold amino acid homologies of its product. Furthermore, the skc gene sequence shows no extended regions homologous to the staphylokinase gene. Upstream from the skc gene, the putative skc promoter and the ribosome-binding site sequence have been identified; downstream from the coding region, inverted repeat sequences thought to function as transcription terminators have been detected.

Amino Acid Sequence↗
PubMed1984

[Conjugational plasmid transfer from A, B and H streptococci to N streptococci].

Plasmid-mediated resistance to erythromycin and chloramphenicol was successfully transferred from group A, B and H streptococci to group N streptococci by a process akin to conjugation. The results showed that plasmids from streptococcal groups other than N were able to replicate in lactic streptococci as well. The transfer experiments were carried out by using a membrane filter mating technique. Four of the five plasmids used (pSM15346, pSM10419, pIP501, and pEL1) were transferred at frequencies ranging from 10(-1) to 10(-8) transconjugants per donor colony-forming unit. The highest transfer frequencies were obtained when S. pyogenes strain 15346 (pSM15346) served as the donor strain. The identy of transconjugants was verified by testing for the presence of unselected markers of the recipient strains, and both transduction and transformation were ruled out as the mechanisms of transfer.

Chloramphenicol↗
PubMed1984

Expression of a streptokinase gene from Streptococcus equisimilis in Streptococcus sanguis.

Using recombinant DNA techniques, we introduced a previously cloned streptokinase gene from Streptococcus equisimilis into the Challis strain of S. sanguis (group H). The gene was expressed in the new host under the control of its own promoter and the gene product had biological properties identical to authentic streptokinase. However, the molecular weight of cloned streptokinase (42 K) as expressed by S. sanguis was substantially lower than that of authentic streptokinase (47 K). Since the cloned streptokinase gene encoded a 47 K mature protein, the lowered molecular weight of S. sanguis streptokinase may reflect posttranslational proteolytic cleavage, which leaves the biological activity of the gene product and its serological reactivity unimpaired.

Cloning, Molecular↗
PubMed1984

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage lambda replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene ( skc ). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.

Cloning, Molecular↗
PubMed1982

Sequence relationships between plasmids associated with conventional MLS resistance and zonal lincomycin resistance in Streptococcus pyogenes.

By using electron microscopy of self-annealed DNA and restriction enzyme analysis, we have compared the physical maps of two group A streptococcal plasmids associated with conventional MLS resistance (pEL1; 20 Md) and zonal lincomycin resistance (pSM10419; 15 Md). Of their monomeric molecules, about 40% and 60%, respectively, are occupied by identical non-tandem inverted repeats containing sequences specifying putative replication functions. Sequence homology also exists between their resistance determinants which are located in unique DNA. Moreover, homology between additional regions of unknown function is so extensive and restriction fragment arrangement so similar that, formally, pSM10419 can be considered a deletion variant of pEL1. The results suggest that MLS and zonal lincomycin resistance have the same biochemical basis (i.e. methylation of 23S ribosomal RNA) and differ only quantitatively in the inducible control systems.

Base Sequence↗