394delTT: a Nordic cystic fibrosis mutation

In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.     

Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Satellite DNA sequences in the New World primateCebus apella (Platyrrhini,Primates)

Two satellite DNAs, designated CapA and CapB, were isolated from the neotropical primate,Cebus apella. The satellites exhibit nonoverlapping distributions onC. apella chromosomes. CapA is a major component of interstitial regions of constitutive heterochromatin, a very large block of heterochromatin comprising most of the long arm of chromosome 11, and some telomeres. The CapA monomer has a length of about 1500 bp and appears recently to have undergone an amplification episode in theC. apella genome. CapA-like sequences are probably present in members of the family Cebidae (to whichC. apella belongs), but not in members of the family Callitrichidae (marmosets). CapB sequences can be detected at the centromeres of manyC. apella chromosomes, and similar sequences are present in all neotropical primates. The 342 bp CapB monomer shares 60%–64% sequence identity with several alpha satellite sequences of human origin. Because of its structure, sequence, and location, it appears that CapB is the New World primate homolog of Old World primate alpha satellite DNA.     

Modulation of SPARC Expression during Butyrate-Induced Terminal Differentiation of Cultured Human Keratinocytes: Regulation via a TGF-β-Dependent Pathway

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC⁺ cells increased with time and peaked immediately postconfluence (31.3 ± 6.3% SPARC⁺). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G₂/M and G₁ phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-β (TGF-β) production since incubation in the presence of NaB + neutralizing antibodies to TGF-β inhibited both the expression of SPARC by 72% and development of mature CEs.