Restriction Fragment Profiles Research Articles

Genetic diversity within Streptococcus mutans evident from chromosomal DNA restriction fragment polymorphisms

Attempts to study the acquisition, transmission, and other aspects of the natural history of Streptococcus mutans infections in humans have been hampered by limitations and inconsistencies in methods by which phenotypic characteristics of individual isolates are examined. Because most mutans streptococci associated with human dental caries fall within the biotype I (serotypes c and f) grouping, designated S. mutans, these typing methods are of little value in distinguishing individual isolates. Here we show that strains of S. mutans obtained from over 30 individuals demonstrate unique "fingerprints" of chromosomal DNA digested with restriction endonuclease HaeIII. To demonstrate that this polymorphism in restriction fragments can be used to study the acquisition and transmission of this organism, we examined isolates of S. mutans from three mother-infant pairs obtained at the time the infant first became colonized by this organism. Results indicate that strains of S. mutans found in infants exhibit restriction fragment profiles identical to those of their mothers, strongly supporting the notion that mothers transmit this organism to their infants. Also, we show that strains of S. mutans with the same restriction fragment profile were stably maintained over a 3-year interval in the one mother-infant pair studied. Moreover, we found that mothers and their infants harbored only a few individual strains, suggesting that transmission of this organism is probably confined within discrete family cohorts. Collectively, these findings demonstrate the potential utility of genomic fingerprinting in studying the natural history of S. mutans infections in humans.

Normal polymorphic variations and transcription of the decay accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells.

In paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic anemia, deficiency of decay accelerating factor (DAF) renders blood cells susceptible to increased deposition of autologous complement activation fragments (C3b) and complemented-mediated injury. To investigate the mechanism of the DAF defect, DNA and mRNA from normal and PNH leukocytes were compared in blot hybridization assays by using DAF cDNA and oligonucleotide probes. Southern analyses of DNA from normal cells revealed a single gene spanning approximately equal to 35 kilobases of DNA. Six HindIII banding patterns were distinguishable among normal individuals. In family studies, the patterns segregated as three homozygous and three heterozygous genotypes deriving from three haplotypes: A, B, and C with frequencies of 0.47, 0.36, and 0.17, respectively. Oligonucleotide mapping localized the polymorphic HindIII sites to two noncoding regions in the vicinity of exons encoding (i) the protein oligosaccharide-rich domain and (ii) the mRNA 3'-untranslated region. Analyses of DNA from DAF-negative leukocytes of eight PNH patients demonstrated restriction fragment profiles identical to those of normal individuals for all enzymes studied. Three patients had the BC (normals = 3/32), three patients had the AA (normals = 6/32), and two patients had the AC (normals = 8/32) HindIII genotype. Of the three PNH patients exhibiting the BC genotype, family studies of two demonstrated the expected inheritance patterns, and RNA gel blot analyses of two showed mRNA transcripts indistinguishable from those in normal cells. The absence of DAF gene or mRNA alterations in affected PNH cells that lack other glycolipid-anchored proteins as well as DAF argues that the lesion underlying PNH cells resides in the glycolipid-anchor pathway.