A comparison of methods for the isolation and fractionation of reticulocyte ribosomes

1. Polysomes, ribosomes and pH5 enzymes were isolated from rabbit reticulocytes by acidifying the post-mitochondrial supernatant to pH6.0 to precipitate all ribonucleoprotein particles and about half the pH5 enzymes; the precipitate was redissolved in buffer, pH7.6, and fractionated by zone centrifuging. 2. The isolation of polysome-rich and ribosome-rich fractions from the post-mitochondrial supernatant was also examined. 3. Studies of the stability of polysomes revealed that dissociation into sub-units occurred when both bound and free Mg(2+) was chelated by EDTA or when the pH was increased above pH8.8.     

The secondary structure of ribosomal ribonucleic acid in solution

1. The u.v.-absorption spectrum of ribosomal RNA from rabbit reticulocytes was studied as a function of temperature at different pH values. The changes in the spectrum over the range 220-320mmu were interpreted on the basis of the assumption that the effect of denaturation and ionization are additive. The results suggest that in neutral salt solutions the secondary structure of the ribosomal RNA samples studied is due to two species of helical segments stabilized principally, if not solely, by complementary base pairs but differing in nucleotide composition: each species appears to be heterogeneous in other respects in view of the breadth of the melting ranges. 2. The number of base pairs per helical segment was estimated to be small (between 4 and 17) on the basis of the relation between melting temperature and chain length previously established by Lipsett and others for model compounds. Small fragments (about 2s) obtained by alkaline hydrolysis appeared to form the same helical segments as the intact molecule in accord with the estimated size of these segments. 3. Specific nucleotide sequences appear necessary to account for the hysteresis observed on titrating ribosomal RNA with acid or alkali within the range pH3.0-7.0 since this phenomenon was less pronounced for Escherichia coli transfer RNA and for RNA from turnip yellow-mosaic virus.     

Interaction of mutations affecting growth rate and resistance to streptomycin in pneumococci and streptococci

Krauss, Marjorie R. (New York University Medical Center, New York, N.Y.), James C. King, and Rody P. Cox. Interaction of mutations affecting growth rate and resistance to streptomycin in pneumococci and streptococci. J. Bacteriol. 92:1337-1344. 1966.-A strain of Streptococcus (Viridans group) was shown by transformation reactions to be the carrier of two interacting mutations. One produced resistance to streptomycin and a slow rate of growth; the only effect of the second was an increase in growth rate when it was added by transformation to streptococcal strains that had already been transformed to bear the first. Similar modifying mutations were observed in strains of streptococci and pneumococci into which the first mutation had been introduced by transformation.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Recombination of 4p16 DNA markers in an unusual family with Huntington disease

The Huntington disease (HD) mutation has been localized to human chromosome 4p16, in a 6-Mb region between the D4S10 locus and the 4p telomere. In a report by Robbins et al., a family was identified in which an affected individual failed to inherit three alleles within the 6-Mb region originating from the parental HD chromosome. To explain these results, it was suggested that the HD locus (HD) lies close to the telomere and that a recombination event took place between HD and the most telomeric marker examined, D4S90. As a test of this telomere hypothesis, we examined six members of this family, five of whom are affected with HD, for the segregation of 12 polymorphic markers from 4p16, including D4S169, which lies within 80 kb of the 4p telomere. We separated, in somatic cell hybrids, the chromosomes 4 from each family member, to determine the phase of marker alleles on each chromosome. We excluded nonpaternity by performing DNA fingerprint analyses on all six family members, and we found no evidence for chromosomal rearrangements when we used high-resolution karyotype analysis. We found that two affected siblings, including one of the patients originally described by Robbins et al., inherited alleles from the non-HD chromosome 4 of their affected parents, throughout the 6-Mb region. We found that a third affected sibling, also studied by Robbins et al., inherited alleles from the HD chromosome 4 of the affected parent, throughout the 6-Mb region. Finally, we found that a fourth sibling, who is likely affected with HD, has both a recombination event within the 6-Mb region and an additional recombination event in a more centromeric region of the short arm of chromosome 4. Our results argue against a telomeric location for HD and suggest that the HD mutation in this family is either associated with DNA predisposed to double recombination and/or gene conversion within the 6-Mb region or is in a gene that is outside this region and that is different from that mutated in most other families with HD.