Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Effect of Moniliformis moniliformis density on distribution within the definitive host population (Rattus norvegicus)

The population dynamics of Moniliformis moniliformis was studied in ‘free-ranging’ laboratory rats, Rattus norvegicus, presented with different relative density levels of M. moniliformis in cockroaches, Periplaneta americana. Changes in selected population parameters of the negative binomial distribution were evaluated as indicators of changes in aggregation. A significant increase in the degree of aggregation of parasites occurred as a result of the increase in relative density of infective stages available to the rats. This increase in aggregation was due to the increase in over-dispersion that occurred in female rats only. The degree of aggregation in females was found to be significantly higher than that in males at both treatment levels. The best indicators of the degree of aggregation were found to be the ratio of the variance to the relative density and the ratio of the log-variance to log-relative density. Changes in k were not correlated with changes in over-dispersion or the relative density.     

Neuropeptide gene expression and neural activity: Assessing a working hypothesis in nucleus caudalis and dorsal horn neurons expressing preproenkephalin and preprodynorphin

1. The working hypothesis that neuropeptide gene expression in a neuron is an indicator of that neuron's physiological activity is discussed. 2. Representative examples from the literature are presented to support the hypothesis. 3. Further, we discuss the regulation of expression of two opioid peptides, preproenkephalin and preprodynorphin, in laminae I and II of the spinal cord and in nucleus caudalis of the trigeminal nuclear complex, where they may play a role in pain modulation. 4. The expression of the opioid peptide genes can be induced by both painful and nonnoxious stimuli in neurons in time-dependent and sensory-specific fashions.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Fast axon activity and the motor pattern in cockroach legs during swimming

Abstract Electromyographic recordings were made from muscles that extend the trochanter/femur of each of the six legs of American cockroaches, Periplaneta americana (L.), while the insects swam in water. The recordings showed two novel features. (1) During swimming, muscle activity in different legs was coordinated in the alternating tripod pattern commonly seen during free walking on land, not in the pattern of synchronous leg pairs common to other large terrestrial insects in water. (2) Fast axons were usually recruited along with slow axons, even when the insect swam at a moderate pace. Fast axon activity always started after the middle of the slow axon burst in intact insects, but vanished from most bursts in the stump of the leg after amputation of the femur. The alternating tripod pattern was maintained even after amputation. Possible causes of fast axon recruitment are discussed.     

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.