Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were able to demonstrate that failure of efficient transfer for the pGKV plasmids was, except for one case, caused by incompatibility of these plasmids with resident plasmids of the recipient strain.     

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

Summary The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

Summary A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the -galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.     

Severe combined immune deficiency due to a homozygous 3.2-kb deletion spanning the promoter and first exon of the adenosine deaminase gene

We have investigated the structural gene for adenosine deaminase (ADA) in a female infant with ADA deficiency associated severe combined immune deficiency (ADA-SCID) disease and her family by DNA restriction-fragment-length analysis. In this family a new ADA-specific restriction-fragment-length variant was detected, which involves a 3.2-kb deletion spanning the ADA promoter as well as the first exon. It was found that the patient, who was born to a consanguineous couple, was homozygous and both her parents and her brother were heterozygous for the deletion. No ADA-specific mRNA could be detected by hybridization in fibroblasts derived from this patient. Thus the patient was established to be homozygous for a true null ADA allele. In the light of the apparently normal development of most tissues except the lymphoid tissue the above finding directly questions the classification of ADA as a 'housekeeping' enzyme.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

Developmental biology of Pneumocystis carinii, and alternative view on the life cycle of the parasite.

In this paper we present, based on elaborate ultrastructural studies, data on the existence of both intracellular and extracellular stages of Pneumocystis carinii, which result in a proposal of a new life cycle of the parasite. Up to now the formation of daughter cells in thick-walled pneumocysts is supposed to be the only way of multiplication. The present study shows that in rats treated with cortisone acetate the formation of daughter cells also takes place within thin-walled pneumocysts. In our opinion this way of multiplication is important for the understanding of the rapid increase in number of the parasites in an infected lung. The presence of pneumocysts inside the alveolar epithelial cells suggests that intracellular development of the parasites can occur, but the method of cell penetration, intracellular multiplication and parasite liberation is still unknown. Moreover our observations for the first time indicate a direct pathogenicity of the parasites in host cells.     

Basic defect in the expression of adenosine deaminase in ADA- SCID disease investigated through the cells of an obligate heterozygote

Summary The nature of the defect of a female baby who died of severe combined immunodeficiency (SCID) disease associated with adenosine deaminase deficiency (ADA^-) was investigated. Since tissue or tissue culture material was not available for subsequent studies, the expression of ADA in her cells was investigated in the somatic cell hybrid clones derived from a fusion between the lymphocytes from one of her two obligate heterozygote parents and thymidine kinase deficient Chinese hamster (a3) fibroblasts. The results of analyses of the human chromosomes and biochemical markers in 12 independent clones and 27 subclones indicated that the ADA deficiency in the patient is determined probably by a mutation in the structural gene for ADA in chromosome 20 leading either to the production of catalytically defective molecules or to the cessation of the production of ADA. Incidentally, the involvement of chromosome 2, which carries a gene for adenosine deaminase complexing protein (ADCP), in the causation of ADA deficiency was excluded. The in vitro approach through the cells from an obligate heterozygote described in this paper may have a general application in pursuing studies on other cases of inborn errors of metabolism whenever the material from the affected individuals (i.e., the homozygotes) is not available or not suitable for direct investigations.A part of this work was presented at the New York State Department of Health, Birth Defects Institute Symposium IX (Inborn Errors of Specific Immunity), Albany, October 16–18, 1978 and reported as an abstract in the proceedings of the Fifth International Conference on Human Gene Mapping, Edinburgh, July 1979. Cytogenet Cell Genet 22:164     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.     

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.