BpuAmI: a novel SacI neoschizomer from Bacillus pumilus discovered in an isolate from Amazon Basin, recognizing 5'-GAG¯CTC-3'

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Abstract

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.

Translated abstract

Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG¯CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.

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Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

S Altschul (1997)

The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.