Isolation and characterization of unusual gin mutants.

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation.     

An experimental method for measuring force on the spinal facet joint: description and application of the method.

A technique is described for measuring load magnitude and resultant load contact location in the facet joint in response to applied loads and moments, and the technique applied to the canine lumbar spine motion segment. Due to the cantilever beam geometry of the cranial articular process, facet joint loads result in surface strains on the lateral aspect of the cranial articular process. Strains were quantified by four strain gages cemented to the bony surface of the process. Strain measured at any one gage depended on the loading site on the articular surface of the caudal facet and on the magnitude of the facet load. Determination of facet loads during in vitro motion segment testing required calibration of the strains to known loads of various magnitudes applied to multiple sites on the caudal facet. The technique is described in detail, including placement of the strain gages. There is good repeatability of strains to applied facet loads and the strains appear independent of load distribution area. Error in the technique depends on the location of the applied facet loads, but is only significant in nonphysiologic locations. The technique was validated by two independent methods in axial torsion. Application of the technique to five in vitro canine L2-3 motion segments testing resulted in facet loads (in newtons, N) of 74+ / -23 N (mean + / -STD) in 2 newton-meter, Nm, extension, to unloaded in flexion. Lateral bending resulted in loads in the right facet of 40+ / -32 N for 1 Nm right lateral bending and 54+ / -29 N for 1 Nm left lateral bending. 4 Nm Torsion with and without 100 N axial compression resulted in facet loads of 92+ / -27 N and 69+ / -19 N, respectively. The technique is applicable to dynamic and in vivo studies.     

G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor

The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

A Mads-Box Homologue in Ustilago Maydis Regulates the Expression of Pheromone-Inducible Genes but Is Nonessential

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and α-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.