Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

Adsorption of DNA by Bacteria and Their Composites with Minerals

Previous studies revealed the thermodynamic properties of DNA adsorption on pure minerals or biomasses; however, there has been little attempt to develop such studies on bacteria–mineral composites. Equilibrium adsorption experiments, attenuated total reflectance Fourier transform infrared spectroscopy, and isothermal titration calorimetry were employed to investigate the adsorption of DNA by Bacillus subtilis, Pseudomonas putida, and their composites with minerals. Similar capacity and affinity were observed for DNA adsorption on two bacterial cells. However, different patterns were found in the adsorption of DNA by bacteria–mineral composites. The Gram-positive bacterium B. subtilis enhanced the adsorption of DNA on its mineral composites compared with their individual components, while the composites of Gram-negative bacterial cells with kaolinite and goethite bound lower amounts of DNA than the predicted values. The thermodynamic parameters and the Fourier transform infrared spectra showed that van der Waals force and hydrogen bonding are responsible for the DNA adsorption on B. subtilis–minerals and P. putida–kaolinite. By contrast, the entropy increases of excluded water rearrangement and dehydration effect play key roles in the interaction between DNA and P. putida–montmorillonite/goethite composites.     

Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.     

The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The application of strand invasion phenomenon,directed by peptide nucleic acid (PNA) and single‐stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV‐W family

The HERV‐W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV‐W–derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin‐1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV‐W members is highly desirable. A peptide nucleic acid (PNA)–mediated technique for the discrimination between multiple sclerosis‐associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis‐associated retrovirus (MSRV) template, shows high selective potential. Single‐stranded DNA binding protein facilitates the PNA‐mediated, sequence‐specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single‐stranded DNA‐specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV‐W env sequences have been evaluated. We believe that PNA/single‐stranded DNA binding protein–based application has the potential to selectively discriminate particular HERV‐W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho‐neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto‐immunologic background (psoriasis and lupus erythematosus).