Intracisternal A-particle genes as movable elements in the mouse genome.

We analyzed two functionally defective mouse kappa light chain gene variants previously shown to contain novel insertions of repetitive DNA in their intervening sequences [Hawley, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. & Hozumi, N. (1982) Proc. Natl. Acad. Sci. USA 79, 7425-7429]. Heteroduplex analysis of the cloned genes shows that the insertions consist of intracisternal A-particle (IAP) genetic elements. Each insertion includes an IAP 5' long terminal repeat (LTR) sequence and extends to a characteristic IAP internal BamHI site where the IAP sequence is interrupted because the mutant genes were cloned from complete BamHI digests of the cellular DNAs. Restriction enzyme mapping indicates that the 5' LTR boundaries of the inserted IAP elements correspond closely to the previously determined rearrangement sites in the mutant genes. The IAP insertions in the two mutants can be distinguished by restriction-site differences and by the fact that one of them contains a deletion that is absent in the other. Nucleotide sequence data are presented for the LTRs of one full-length IAP gene copy randomly selected from a mouse genomic DNA library. These LTRs show many features typical of known integrated retroviral terminal repeat units, and the entire gene is bracketed by short direct repeats within the adjacent cellular DNA. Thus, the findings show that IAP genetic elements can appear in new locations in mouse cellular DNA and suggest that this may occur through a process of proviral insertion.     

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.     

Pressure-flow and pressure-volume relations in the entire pulmonary vascular bed of the dog determined by two-port analysis.

To quantify the resistance and particularly the capacitance properties of the entire pulmonary vascular bed, isolated perfused lungs of nine dogs were studied. In each dog, with a fixed endotracheal pressure of 5 mm Hg, the arterial pressure-flow and pressure-volume relations were determined while venous pressure was fixed at constant values of 2, 5, and 8 mm Hg. In the same dogs, the venous pressure-flow and pressure-volume relations were also obtained when arterial pressure was fixed at constant values of 15 and 20 mm Hg. The arterial and venous pressure-flow relations could be regarded as linear around the physiological ranges of arterial and venous pressures; however, at very low pressures, these relations became nonlinear. The arterial and venous pressure-volume relations were also approximately linear within the physiological pressure ranges. The mean arterial compliance was 0.1798 ml/mm Hg kg-1 and was independent of venous pressures. The venous compliance values were 0.1236 and 0.0955 ml/mm Hg kg-1 for arterial pressures of 15 and 20 mm Hg, respectively. The sums of the arterial and venous compliances were 0.3034 and 0.2753 ml/mm Hg kg-1 for arterial pressures of 15 and 20 mm Hg, respectively. These values were nearly identical to the mean total compliance, 0.3265 ml/mm Hg kg-1, measured in a separate series of experiments in the same dogs. Therefore, the data obtained in these experiments using two-port analysis techniques represent the compliances and the resistance of the entire pulmonary vascular bed around the normal operating pressures and flows.     

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing

Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10⁻³ per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.     

Intramolecular recombination of chloroplast genome mediated by short direct-repeat sequences in wheat species.

Structural alterations of the chloroplast genome tend to occur at "hot spots" on the physical map. To clarify the mechanism of mutation of chloroplast genome structure in higher plants, we determined the nucleotide sequence of the hot-spot region of chloroplast DNAs related to length mutations (deletions/insertions) in Triticum (wheat) and Aegilops. From a comparison of this region in wheat with the corresponding region of tobacco or liverwort, it is evident that one of the open reading frames in tobacco (ORF512) has been replaced in wheat by the rpl23 gene, which is a member of the ribosomal protein gene operon. In the deleted positions and in the original genome of Triticum and Aegilops, consensus sequences forming short direct repeats were found, indicating that these deletions were a result of intramolecular recombination mediated by these short direct-repeat sequences. By two independent recombination events in the Aegilops crassa type of chloroplast genome, which is shared by Triticum monococcum, Ae. bicornis, Ae. sharonensis, Ae. comosa, and Ae. mutica, the novel chloroplast DNA sequences of T. aestivum and Ae. squarrosa were generated. This finding indicates the existence of illegitimate recombination in the chloroplast genome and presents a mechanism for producing genetic diversity of that genome.     

Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.     

Characterization of subtype A HIV-1 from Africa by full genome sequencing.

OBJECTIVE: To improve our understanding of the genetic complexity of HIV-1 subtype A by increasing the number of subtype A isolates that have been sequenced in their entirety. METHODS: Nine HIV-1-seropositive patients from Africa living in Sweden contributed peripheral blood mononuclear cells (PBMC) for this study. Sequencing of the C2-V3 region of env had shown them to be subtype A. DNA from virus cultures was used for the amplification of virtually full-length proviral sequences, and the resulting fragment was sequenced. RESULTS: Six of the nine viral isolates were subtype A throughout the genome, or non-recombinant, and all of these were from east Africa. One virus from the Ivory Coast had the AG(IbNG) genetic form, a recombinant form common in west Africa. Two of the isolates were novel recombinants: one was an A/C recombinant and the other was A/D. Analysis of gag reveals three subclusters within the A subtype: one containing the AG(IbNG) subtype viruses, one containing the AE(CM240) viruses and one containing the non-recombinant A viruses. These genetic clusters have different geographical distributions in Africa. CONCLUSION: The prevailing view of HIV-1 subtype A forming a uniform band across the center of sub-Saharan Africa needs revision. In all probability, the most common subtype in west Africa and west central Africa is the AG recombinant, AG(IbNG), whereas in east central Africa it is the non-recombinant subtype A.     

The pattern of mutational involvement of RAS genes in human hematologic malignancies determined by DNA amplification and direct sequencing

DNA from 161 patients with various forms of hematologic malignancies were investigated for mutations in exons 1 and 2 of the N-RAS, K-RAS and Ha-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Mutations involving either codons 11, 12, or 13 of the N-RAS gene were identified in 18 of the 161 patients. The relative frequencies of N-RAS gene mutations in these hematologic disorders was as follows: acute myelogenous leukemia (AML), 15%; acute lymphoblastic leukemia (ALL), 14%; myelodysplastic syndromes, 24%; and myeloid and lymphoid blast crisis of chronic myelogenous leukemia (CML), 3%. No correlation was observed between the presence of mutations and cytologic features or immunophenotype of these malignancies. Mutations involving codons 12 or 13 were equally prevalent, with a glycine to aspartic acid substitution being the most frequently encountered change. A single T-ALL case had a codon 11 mutation resulting in substitution of alanine with threonine. We failed to find mutations in exons 1 and 2 of the K-RAS or Ha-RAS genes in any case except a single AML with a mutation in codon 61 of the K-RAS gene. Also, no mutations were identified in chronic phase of CML, chronic lymphocytic leukemia. Ph1 positive ALL, non-Hodgkin's lymphoma, Hodgkin's disease, or multiple myeloma. These results indicate that RAS mutations, especially those involving exon 1 of the N-RAS gene, are frequent only in a subset of hematologic malignancies.     

Tracing the origin of Brazilian HTLV-1 as determined by analysis of host and viral genes

We compared the genetic diversity of the Brazilian human T-cell lymphotropic virus type 1 isolates with those found in KwaZulu-Natal (KZN), South Africa, and with the genetic background of the hosts. The seroprevalence rate in KZN was 1.7%. All sequences belonged to the A subgroup. The presence of South African sequences in two different clusters from Brazil, and the finding of the beta-globin haplotype in infected hosts are consistent with the transmission of this virus from southern Africa to Brazil.     

Structural rearrangements of the chloroplast genome provide an important phylogenetic link in ferns.

The chloroplast genome of most land plants is highly conserved. In contrast, physical and gene mapping studies have revealed a highly rearranged chloroplast genome in species representing four families of ferns. In all four, there has been a rare duplication of the psbA gene and the order of the psbA, 16S, and 23S rRNA genes has been inverted. Our analysis shows that the described rearrangement results from a minimum of two inversions within the inverted repeat. This chloroplast DNA structure provides unambiguous evidence that phylogenetically links families of ferns once thought to belong to different major evolutionary lineages.     

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

The bulk of late virus-specific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of poloma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the HindII-1 fragment of polyoma DNA, which contains 91% of the viral genome. Hybrid molecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in HindII-1), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.     

Tandemly repeated nonribosomal DNA sequences in the chloroplast genome of an Acetabularia mediterranea strain.

A purified chloroplast fraction was prepared from caps of the giant unicellular green alga Acetabularia mediterranea (strain 17). High molecular weight DNA obtained from these chloroplasts contains at least five copies of a 10-kilobase-pair (kbp) sequence tandemly arranged. This unique sequence is present in DNA from chloroplasts of all stages of the life cycle examined. A chloroplast rDNA clone from mustard hybridized with some restriction fragments from Acetabularia chloroplast DNA but not with the repeated sequence. An 8-kbp EcoRI-Pst I fragment of the repeated sequence was cloned into pBR322 and used as a hybridization probe. No homology was found between the cloned 8-kbp sequence and chloroplast DNA from related species Acetabularia crenulata or chloroplast DNA from spinach.     

Architecture of the Escherichia coli ribosome as determined by immune electron microscopy.

Binding sites for antibodies specific to nineteen of the twenty-one ribosomal proteins from the 30S subunit of E. coli ribosomes have been localized on the surface of the 30S ribosomal subunit by immune electron microscopy. The locations of 13 ribosomal proteins from the 50S subunit were similarily assessed. The arrangement of these proteins is illustrated in three-dimensional models of the 30S and 50S ribosomal subunits and of 70S ribosomes. With specific antibodies to six proteins of the 30S subunit we found only one attachment point for each protein. Antibodies against each of nine of the proteins attached at two separate sites that were separated by various distances. Four further proteins were exposed at three or four sites for antibody binding. Altogether eight to ten of the 19 proteins of the 30S subunit have shown antibody attachment sites at remote points on the surface of the ribosome, at distances which are incompatible with globular shapes; these proteins must therefore have elongated or fibrous structures within the ribosome. On the other hand, only two proteins of the 50S subunit, namely L11 and L18, have so far revealed two separated antibody binding sites; proteins L7/L12 occurred, however, at multiple sites.     

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype‐specific PCR and deep sequencing

The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct‐acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR‐based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8%; however, this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (P < .05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR next‐generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the nonquantitative nature of the technique and despite the designation of false‐positive thresholds based on negative controls.     

The massive mitochondrial genome of the angiosperm Silene noctiflora is evolving by gain or loss of entire chromosomes

Across eukaryotes, mitochondria exhibit staggering diversity in genomic architecture, including the repeated evolution of multichromosomal structures. Unlike in the nucleus, where mitosis and meiosis ensure faithful transmission of chromosomes, the mechanisms of inheritance in fragmented mitochondrial genomes remain mysterious. Multichromosomal mitochondrial genomes have recently been found in multiple species of flowering plants, including Silene noctiflora, which harbors an unusually large and complex mitochondrial genome with more than 50 circular-mapping chromosomes totaling ∼7 Mb in size. To determine the extent to which such genomes are stably maintained, we analyzed intraspecific variation in the mitochondrial genome of S. noctiflora. Complete genomes from two populations revealed a high degree of similarity in the sequence, structure, and relative abundance of mitochondrial chromosomes. For example, there are no inversions between the genomes, and there are only nine SNPs in 25 kb of protein-coding sequence. Remarkably, however, these genomes differ in the presence or absence of 19 entire chromosomes, all of which lack any identifiable genes or contain only duplicate gene copies. Thus, these mitochondrial genomes retain a full gene complement but carry a highly variable set of chromosomes that are filled with presumably dispensable sequence. In S. noctiflora, conventional mechanisms of mitochondrial sequence divergence are being outstripped by an apparently nonadaptive process of whole-chromosome gain/loss, highlighting the inherent challenge in maintaining a fragmented genome. We discuss the implications of these findings in relation to the question of why mitochondria, more so than plastids and bacterial endosymbionts, are prone to the repeated evolution of multichromosomal genomes.     

Myocardial tissue recruitment in the dog as determined by double tracer dilution method.

The amount of tissue perfused, as determined from the difference in volume of distribution between a diffusible indicator (125I-antipyrine) and an intravascular indicator (131 I-albumin) was measured at different values of coronary flow, perfusion pressure, and vasomotor tone in the working left ventricle of an open-chest dog. Coronary pressure and flow were regulated independently from the systemic circulation and coronary vasomotor tone was reduced by dipyridamole. At each flow vasomotor tone was assessed by using as a reference the maximal vasodilation induced by arrest of flow. Measured tissue space was considered to be related to the capillary surface area available for tracer diffusion and therefore to the number of perfused capillaries per volume of muscle. A relationship between coronary blood flow and tissue volume was observed. It was found to be independent of vasomotor tone. Vasodilation was found to increase available exchanging capillary surface at a constant perfusion pressure.