Comparison of Phenotypic and Genotypic Methods Used for the Species Identification of Lactobacillus NP51 and Development of a Strain-Specific PCR Assay.

The species level identity of Lactobacillus NP51, a commercial direct-fed microbial previously identified as Lactobacillus acidophilus NP51, was re-evaluated to determine whether new technologies resulted in changes in the original identification. The phenotypic methods for species identification included API 50 CHL kit and two automated systems, Vitek 2 and MIDI (FAME analysis; a total of three independent FAME analyses). Discrepancies among the identification results with all methods of phenotypic analysis were reported. MicroSeqID 500 16S rRNA system (SeqWright Inc., Houston, TX), a genotypic method, identified the organism as Lactobacillus animalis. Cloning, sequencing and subsequent sequence comparison of NP51 16S-23S intergenic spacer region (ISRs) to nucleotide sequence databases using the BLAST search tool indicated that NP51 can now be named L. animalis. When NP51 was originally identified as L. acidophilus, the designation of L. animalis did not exist taxonomically. The NP51 sequence comparisons using BLAST also revealed that NP51 and a strain previously identified as L. animalis LA51 HOFG1 by Flint and Angert are identical strains under different names. A strain-specific primer pair was also identified for HOFG1 by the same research group. A primer pair (using HOFG1 forward pair) also produced an amplicon unique to NP51. These methods demonstrate the significance of genetic-based detection methods both for scientific identification of organisms from biological samples and to prevent misidentification in food and health industry related microorganisms in which proprietary considerations are an important concern.

The gene for a variant form of the polyadenylation protein CstF-64 is on chromosome 19 and is expressed in pachytene spermatocytes in mice.

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999) Proc. Natl. Acad Sci. U. S. A. 96, 6763-6768). Previously, we demonstrated the presence of two distinct forms of the M(r) 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somatic M(r) 64,000 form and a variant M(r) 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form "tau CstF-64," and we describe here the cloning and characterization of the mouse tauCstF-64 cDNA, which maps to chromosome 19. The mouse tauCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with tauCstF-64 from testis. Features of mtauCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro --> Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.