Restriction of bacteriophage lambda by Escherichia coli K

Derivatives of phage lambda, for which the numbers and positions of the recognition sites for endonuclease R. Eco_k are known, were used as substrates for the Escherichia coli K restriction system in vivo and in vitro. A single unmodified recognition site was sufficient for a DNA molecule to be bound and broken by the K restriction enzyme. Although discrete fragments of DNA were not produced, the breaks were made preferentially in the proximity of the recognition site. Breakage of a DNA molecule with only one recognition site required a 10 to 40-fold higher concentration of restriction enzyme than breakage of a DNA molecule with two or more recognition sites, but these substrates were all equally effective in a binding assay for the enzyme.The polynucleotide kinase reaction provided no evidence for new 5′-terminal sequences generated by restriction in vitro; the 5′ termini were either refractory to the polynucleotide kinase reaction or had no sequence specificity.     

Circadian changes in prolactin cell activity in the pituitary of the teleost Poecilia latipinna in freshwater

Summary Quantitative morphometric studies with the electron microscope were made on the prolactin cells of wild freshwater sailfin mollies taken in Florida in August at four different times of day. The results indicate a circadian rhythm in the prolactin cell, the period of highest synthetic activity being from midday to evening, as indicated by the condition of nucleus, Golgi apparatus and rough endoplasmic reticulum, and by the incidence of granule release profiles. No circadian changes were detected in the ACTH cells.Several distinct sites of prolactin granule release were recognised. However, there was no conclusive evidence of granule release by any mechanism other than classical exocytosis.We thank Mr. W. Thomson for technical assistance and Ann Grier for help in collecting the fish. T.F.C. Batten is in receipt of an S.R.C. Research Studentship.     

In vivo fitness costs of different Gag CD8 T-cell escape mutant simian-human immunodeficiency viruses for macaques

The kinetics of immune escape and reversion depend upon the efficiency of CD8 cytotoxic T lymphocytes (CTL) and the fitness cost of escape mutations. Escape kinetics of three simian immunodeficiency virus Gag CTL epitopes in pigtail macaques were variable; those of KP9 and AF9 were faster than those of KW9. Kinetics of reversion of escape mutant virus to wild type upon passage to na?ve major histocompatibility complex-mismatched macaques also varied. Rapid reversion occurred at KP9, gradual biphasic reversion occurred at AF9, and escape mutant KW9 virus failed to revert. The fitness impact of these mutations is KP9 > AF9 > KW9. These data provide insights into the differential utility of CTL in controlling viremia.     

Decreasing initial telomere length in humans intergenerationally understates age‐associated telomere shortening

Telomere length shortens with aging, and short telomeres have been linked to a wide variety of pathologies. Previous studies suggested a discrepancy in age‐associated telomere shortening rate estimated by cross‐sectional studies versus the rate measured in longitudinal studies, indicating a potential bias in cross‐sectional estimates. Intergenerational changes in initial telomere length, such as that predicted by the previously described effect of a father's age at birth of his offspring (FAB), could explain the discrepancy in shortening rate measurements. We evaluated whether changes occur in initial telomere length over multiple generations in three large datasets and identified paternal birth year (PBY) as a variable that reconciles the difference between longitudinal and cross‐sectional measurements. We also clarify the association between FAB and offspring telomere length, demonstrating that this effect is substantially larger than reported in the past. These results indicate the presence of a downward secular trend in telomere length at birth over generational time with potential public health implications.     

Development of methods for quantitative comparison of pooled shRNAs by mass sequencing

Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.     

Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides

Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.     

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.