RegA proteins from phage T4 and RB69 have conserved helix–loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ∼7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.

Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.

Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors

The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-γ-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.

The use of mRNA display to select high-affinity protein-binding peptides

We report the use of “mRNA display,” an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of ≈1013 random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibody–antigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

Assessment of Customer Service in Academic Health Care Libraries (ACSAHL): an instrument for measuring customer service*†

Objectives: In a pilot study, the library had good results using SERVQUAL, a respected and often-used instrument for measuring customer satisfaction. The SERVQUAL instrument itself, however, received some serious and well-founded criticism from the respondents to our survey. The purpose of this study was to test the comparability of the results of SERVQUAL with a revised and shortened instrument modeled on SERVQUAL. The revised instrument, the Assessment of Customer Service in Academic Health Care Libraries (ACSAHL), was designed to better assess customer service in academic health care libraries.

Health sciences libraries in Kuwait: a study of their resources, facilities, and services

The purpose of this study was to examine the current status of health sciences libraries in Kuwait in terms of their staff, collections, facilities, use of information technology, information services, and cooperation. Seventeen libraries participated in the study. Results show that the majority of health sciences libraries were established during the 1980s. Their collections are relatively small. The majority of their staff is nonprofessional. The majority of libraries provide only basic information services. Cooperation among libraries is limited. Survey results also indicate that a significant number of health sciences libraries are not automated. Some recommendations for the improvement of existing resources, facilities, and services are made.

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes.

Providing health information to the general public: a survey of current practices in academic health sciences libraries*

A questionnaire was mailed to 148 publicly and privately supported academic health sciences libraries affiliated with Association of American Medical Colleges (AAMC)–accredited medical schools in the United States and Canada to determine level of access and services provided to the general public. For purposes of this study, “general public” was defined as nonaffiliated students or health care professionals, attorneys and other nonhealth-related professionals, patients from affiliated or other hospitals or clinics, and general consumers. One hundred five (71%) libraries responded. Results showed 98% of publicly supported libraries and 88% of privately supported libraries provided access to some or all of the general public. Publicly supported libraries saw greater numbers of public patrons, often provided more services, and were more likely to circulate materials from their collections than were privately supported libraries. A significant number of academic health sciences libraries housed a collection of consumer-oriented materials and many provided some level of document delivery service, usually for a fee. Most allowed the public to use some or all library computers. Results of this study indicated that academic health sciences libraries played a significant role in serving the information-seeking public and suggested a need to develop written policies or guidelines covering the services that will be provided to minimize the impact of this service on primary clientele.

Standards for vision science libraries

The minimum levels of staffing, services, budget, and technology that should be provided by a library specializing in vision science are presented. The scope and coverage of the collection is described as well. These standards may be used by institutions establishing libraries or by accrediting bodies reviewing existing libraries.

Interlibrary loan in primary access libraries: challenging the traditional view

Introduction: Primary access libraries serve as the foundation of the National Network of Libraries of Medicine (NN/LM) interlibrary loan (ILL) hierarchy, yet few published reports directly address the important role these libraries play in the ILL system. This may reflect the traditional view that small, primary access libraries are largely users of ILL, rather than important contributors to the effectiveness and efficiency of the national ILL system.