Henry V. Huang

A single VH gene segment encodes the immune response to phosphorylcholine: Somatic mutation is correlated with the class of the antibody

Abstract The immune response in BALB/c mice to phosphorylcholine is highly restricted in its heterogeneity. Of the 19 immunoglobulins binding phosphorylcholine for which complete V H -segment amino acid sequences have been determined, 10 employ a single sequence, denoted T15 after the prototype V H sequence of this group of antibodies. The remaining 9 of these V H segments are variants differing by 1 to 8 residues from the T15 sequence. Using a cloned V H cDNA probe complementary to the T15 sequence, we isolated from a mouse sperm genomic library clones corresponding to four V H gene segments that by DNA sequence analysis are >85% homologous to one another. These four V H gene segments have been denoted the T15 V H gene family. These V H gene segments are most, if not all, of the germline V H gene segments that could encode the V H sequences of antibodies that bind phosphorylcholine. One of these four genes contains the T15-V H -coding sequence. When the T15-family V H gene segments were compared with the complete V H protein sequences of 19 hybridoma and myeloma immunoglobulins that bind phosphorylcholine, several striking conclusions could be drawn. First, all of these V H regions must have arisen from the germline T15 V H gene segment. Thus virtually the entire immune response to phosphorylcholine is derived from a single V H -coding sequence. Nine of the 19 V H regions were variants differing from the T15-V H -coding sequence and, accordingly, must have arisen by a mechanism of somatic diversification. Second, the variants appear to be generated by a somatic mutation mechanism. They cannot be explained by recombination or gene conversion among members of the T15 gene family. Third, somatic mutation is correlated with the class of the antibody. All of the somatic variation is found in the V H regions derived from antibodies of the IgA and IgG classes. The IgM molecules express the germline T15 V H gene segment exclusively.

Initial review of methods for cascading failure analysis in electric power transmission systems IEEE PES CAMS task force on understanding, prediction, mitigation and restoration of cascading failures

Large blackouts are typically caused by cascading failure propagating through a power system by means of a variety of processes. Because of the wide range of time scales, multiple interacting processes, and the huge number of possible interactions, the simulation and analysis of cascading blackouts is extremely complicated. This paper defines cascading failure for blackouts and gives an initial review of the current understanding, industrial tools, and the challenges and emerging methods of analysis and simulation.

Association of the sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

A Positive Feedback Loop Promotes Transcription Surge That Jump-Starts Salmonella Virulence Circuit

The PhoP/PhoQ two-component system is a master regulator of Salmonella pathogenicity. Here we report that induction of the PhoP/PhoQ system results in an initial surge of PhoP phosphorylation; the occupancy of target promoters by the PhoP protein; and the transcription of PhoP-activated genes, which then subsides to reach new steady-state levels. This surge in PhoP activity is due to PhoP positively activating its own transcription, because a strain constitutively expressing the PhoP protein attained steady-state levels of activation asymptotically, without the surge. The strain constitutively expressing the PhoP protein was attenuated for virulence in mice, demonstrating that the surge conferred by PhoPs positive feedback loop is necessary to jump-start Salmonellas virulence program.

Effect of base pair mismatches on recombination via the RecBCD pathway

SummaryThe effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches. Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd number of crossovers. In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold. These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair. Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2-or 12-fold, depending on the location of the mismatch. However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts. Thus a single match repair seems unable to act on single mismatches in very short homologies during recombination.

Conflicting needs for a Salmonella hypervirulence gene in host and non‐host environments

The Gram‐negative pathogen Salmonella enterica harbours a periplasmic D‐Ala‐D‐Ala dipeptidase (termed PcgL), which confers the ability to grow on D‐Ala‐D‐Ala as sole carbon source. We now demonstrate that inactivation of the pcgL gene renders Salmonella hypervirulent. This phenotype results from the accumulation of peptidoglycan‐derived D‐Ala‐D‐Ala in the pcgL mutant and not from an intrinsically faster growth rate. Synthetic D‐Ala‐D‐Ala (but not L‐Ala‐L‐Ala or D‐Ala) increased the number of wild‐type Salmonella in the liver and spleen of mice within 24 h of injection, suggesting that D‐Ala‐D‐Ala interferes with some aspect of innate immunity. However, the pcgL mutant was unable to grow on D‐Ala‐D‐Ala as sole carbon source and was defective for survival in nutrient‐poor conditions. We identified clinical isolates lacking D‐Ala‐D‐Ala dipeptidase activity and unable to grow on D‐Ala‐D‐Ala because of inactivation of the pcgL gene. Our data suggest that genes (such as pcgL) that, when mutated make pathogens more virulent, may be retained because their contribution to pathogen fitness in non‐host environments outweighs potential advantages of the hypervirulent vari‐ant in the infected host.

Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales

ABSTRACT Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.

Analysis of differentially-regulated genes within a regulatory network by GPS genome navigation

MOTIVATION A critical challenge of the post-genomic era is to understand how genes are differentially regulated even when they belong to a given network. Because the fundamental mechanism controlling gene expression operates at the level of transcription initiation, computational techniques have been developed that identify cis regulatory features and map such features into expression patterns to classify genes into distinct networks. However, these methods are not focused on distinguishing between differentially regulated genes within a given network. Here we describe an unsupervised machine learning method, termed GPS for gene promoter scan, that discriminates among co-regulated promoters by simultaneously considering both cis-acting regulatory features and gene expression. GPS is particularly useful for knowledge discovery in environments with reduced datasets and high levels of uncertainty. RESULTS Application of this method to the enteric bacteria Escherichia coli and Salmonella enterica uncovered novel members, as well as regulatory interactions in the regulon controlled by the PhoP protein that were not discovered using previous approaches. The predictions made by GPS were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, and is a central element in a highly connected network. AVAILABILITY The scripts and programs used in this work are accessible from the gps-tools.wustl.edu website. Data and predictions are available by request.

Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site. Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

The promoter architectural landscape of the Salmonella PhoP regulon

The DNA‐binding protein PhoP controls virulence and Mg2+ homeostasis in the Gram‐negative pathogen Salmonella enterica serovar Typhimurium. PhoP regulates expression of a large number of genes that differ both in their ancestry and in the biochemical functions and physiological roles of the encoded products. This suggests that PhoP‐regulated genes are differentially expressed. To understand how a bacterial activator might generate varied gene expression behaviour, we investigated the cis‐acting promoter features (i.e. the number of PhoP binding sites, as well as their orientation and location with respect to the sites bound by RNA polymerase and the sequences that constitute the PhoP binding sites) in 23 PhoP‐activated promoters. Our results show that natural PhoP‐activated promoters utilize only a limited number of combinations of cis‐acting features – or promoter architectures. We determine that PhoP activates transcription by different mechanisms, and that ancestral and horizontally acquired PhoP‐activated genes have distinct promoter architectures.