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It is our belief that large-scale DNA sequencing will revolutionize the study of molecular biology, developmental biology, genetics, and evolution for the TCR gene families and, indeed, all multigene families. We also stress the power of comparative analyses for large-scale DNA sequencing efforts.

The variable region-containing chitin-binding proteins (VCBPs) are found in protochordates and consist of two tandem immunoglobulin variable (V)-type domains and a chitin-binding domain. We previously have shown that these polymorphic genes, which primarily are expressed in the gut, exhibit characteristics of immune genes. In this report, we describe VCBP genomic organization and characterize adjacent and intervening genetic features which may influence both their polymorphism and complex transcriptional repertoire., VCBP genes 1, 2, 4, and 5 are encoded in a single contiguous gene-rich chromosomal region and VCBP3 is encoded in a separate locus. The VCBPs exhibit extensive haplotype variation, including copy number variation (CNV), indel polymorphism and a markedly elevated variation in repeat type and density. In at least one haplotype, inverted repeats occur more frequently than elsewhere in the genome. Multi-animal cDNA screening, as well as transcriptional profilingusing a novel transfection system, suggests that haplotype-specific transcriptional variants may contribute to VCBP genetic diversity., The availability of the Branchiostoma floridae genome (Joint Genome Institute, Brafl1), along with BAC and PAC screening and sequencing described here, reveal that the relatively limited number of VCBP genes present in the amphioxus genome exhibit exceptionally high haplotype variation. These VCBP haplotypes contribute a diverse pool of allelic variants, which includes gene copy number variation, pseudogenes, and other polymorphisms, while contributing secondary effects on gene transcription as well.

The runt domain transcription factors are key regulators of developmental processes in bilaterians, involved both in cell proliferation and differentiation, and their disruption usually leads to disease. Three runt domain genes have been described in each vertebrate genome (the RUNX gene family), but only one in other chordates. Therefore, the common ancestor of vertebrates has been thought to have had a single runt domain gene. Analysis of the genome draft of the fugu pufferfish (Takifugu rubripes) reveals the existence of a fourth runt domain gene, FrRUNT, in addition to the orthologs of human RUNX1, RUNX2 and RUNX3. The tiny FrRUNT packs six exons and two putative promoters in just 3 kb of genomic sequence. The first exon is located within an intron of FrSUPT3H, the ortholog of human SUPT3H, and the first exon of FrSUPT3H resides within the first intron of FrRUNT. The two gene structures are therefore "interlocked". In the human genome, SUPT3H is instead interlocked with RUNX2. FrRUNT has no detectable ortholog in the genomes of mammals, birds or amphibians. We consider alternative explanations for an apparent contradiction between the phylogenetic data and the comparison of the genomic neighborhoods of human and fugu runt domain genes. We hypothesize that an ancient RUNT locus was lost in the tetrapod lineage, together with FrFSTL6, a member of a novel family of follistatin-like genes. Our results suggest that the runt domain family may have started expanding in chordates much earlier than previously thought, and exemplify the importance of detailed analysis of whole-genome draft sequence to provide new insights into gene evolution.

The open data release policy adopted by the large-scale DNA sequencing centers has made accessible valuable information that facilitates research. Herein, we argue that the data producers' rights to receive credit for at least some portion of the analyses of the data must be protected. We suggest that this protection take the form of a specification of the probable content of the primary paper the data producers intend to publish when the data gathering is complete. Rights to publish that paper ought then be restricted to the producers unless they give permission otherwise.

With phage G4 DNA as template, primase (the dnaG protein) synthesizes a 26- to 29-residue RNA transcript at the origin of replication. The sequence starts with ATP and contains a hairpin region of one A-U and seven G-C base pairs. Covalent linkage between the RNA and the newly synthesized complementary DNA chain indicates that the RNA transcript serves as a primer.

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase.

Recombinant lambda phages containing the genes for dnaZ protein (the gamma subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichia coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.

The 29-residue ribonucleotide primer formed by primase at the origin of phage G4 DNA replication (Bouché, J.-P, Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 765-769) was shorter in the presence of deoxynucleoside triphosphates (dNTPs). At 50 micrometer dNTPs and 20 micrometer rNTPs, RNA trancripts no longer than 6 residues were synthesized and these were still effective in priming replication by the DNA polymerase III holoenzyme. Primer synthesis was initiated with ATP; adenosine 5'-O-(3-thiotriphosphate) (Appp(S)), adenosine 5'-tetraphosphate, adenylyl imidodiphosphate (App(NH)p), and ADP were able to substitute for ATP. dATP and GTP were ineffective in initiating replication. DNA replication was stimulated by GTP, suggesting that incorporation of this nucleotide into the second position of the primer trancript by primase produces a more efficient primer. Each of the dNTPs can be incorporated into a hybrid ribonucleotide-deoxyribonucleotide transcript, indicating that primase is able to add either a ribonucleotide or deoxyribonucleotide to the 3'-OH of either of ribo residue or a deoxy residue of the primer terminus. Incorporation of an individual dNTP was less efficient than that of the corresponding rNTP, and the presence of all four dNTPs profoundly depressed RNA synthesis by primase.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.

The genes for the RNA polymerase sigma subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into lambda vectors. Combined restriction, deletion and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into lambda, contains rpoD and at least part of dnaG.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk-factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data, and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific autoantibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies., By correlating patient symptoms with in-depth profiling of blood cells and plasma components throughout COVID-19 infection, this study identifies factors that may predict sustained disease.