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PubMed1976

Size differences among immunoglobulin heavy chains from phosphorylcholine-binding proteins.

The entire sequences of the heavy chain variable regions of M167 and TEPC 15 (phosphorylcholine-binding myeloma proteins of BALB/c origin) have been determined. These sequences are compared with the phosphorylcholine-binding protein M603. T15 differs from M603 at four positions, all of which are located in antigen-binding complementarity regions. M167, in addition to having differences in the complementarity regions, also has five substitutions in the conserved framework portion of the variable region when compared to T15 and M603. Each of the three proteins has a different length in the third complementarity region. It is unlikely that complementarity regions of different lengths associated with similar framework regions could be generated by proposed mechanisms of somatic mutation which are generally limited to point mutations. It appears more likely that these products are directly encoded by different structural germ line genes.

Amino Acid Sequence↗
PubMed2026

Efficacy of the NMIC-150 system in identifying extended-spectrum beta-lactamases in clinical isolates.

Extended-spectrum beta-lactamases (ESBLs) are significant contributors to the growing global crisis of antimicrobial resistance. This study evaluated the performance of the NMIC-150 System for susceptibility testing of third-generation cephalosporins (3GCs) and assessed whether ceftazidime-avibactam and aztreonam-avibactam could identify ESBL-producing carbapenem-resistant Enterobacterales (CREs). A total of 278 non-duplicate clinical isolates (Klebsiella pneumoniae, E. coli, and Proteus mirabilis) were analyzed. Antimicrobial susceptibility was determined using reference broth microdilution (BMD) and the NMIC-150 System. ESBL production was defined as an ≥eight-fold reduction in the minimum inhibitory concentration (MIC) of 3GCs in the presence of clavulanic acid, according to CLSI criteria. Whole-genome sequencing was performed to characterize ESBL and carbapenemase genes among 3GC-resistant isolates. A Random Forest model was used to predict ESBL-producing isolates based on MIC values. The NMIC-150 System demonstrated over 90% categorical and essential agreement with BMD for ceftazidime and ceftriaxone, along with robust predictive performance via Random Forest analysis. These findings suggest that the NMIC-150 System is a reliable platform for 3GC susceptibility testing and that an ≥eight-fold MIC reduction with ceftazidime-avibactam or aztreonam-avibactam may serve as a phenotypic indicator of ESBL production in CRE isolates. In conclusion, the NMIC-150 System shows potential for routine antimicrobial resistance surveillance and may facilitate the rapid identification of ESBL-producing CREs in clinical settings.

Microbial Sensitivity Tests↗
PubMed1977

Structure of hepatitis B Dane particle DNA and nature of the endogenous DNA polymerase reaction.

The circular DNA of hepatitis B Dane particles, which serves as the primer/template for an endogenous DNA polymerase, was analyzed by electrophoresis before and after a polymerase reaction and after digestion by restriction endonuclease or single-strand-specific endonuclease S1. The unreacted molecules extracted from the particles were electrophoretically heterogeneous, and treatment with S1 nuclease produced double-stranded linear DNA ranging in length from 1,700 to 2,800 base pairs (bp). After an endogenous DNA polymerase reaction, two discrete species of DNA molecules were found: a circular form and a linear form 3,200 bp long. The reaction resulted in a population of molecules with an elongated and more homogeneous double-stranded region. These results suggest that the circular molecules in Dane particles have single-stranded regions of varying lengths that are made double stranded during the DNA polymerase reaction. The endogenous DNA polymerase was found to initiate apparently at random in a region spanning more than a third of the molecule. Analysis of restriction endonuclease cleavage fragments of the fully elongated DNA revealed that although the molecules were of a uniform length, they were somewhat heterogeneous in sequence. The sum of the sizes of the 10 major endonuclease Hae III-generated fragments, detected by ethidium bromide, was 3,880 bp. Two additional fragments (B and G) detected by autoradiography after an endogenous DNA polymerase reaction with (32)P-labeled deoxynucleoside triphosphates made the total 4,910 bp.

DNA Restriction Enzymes↗
PubMed2023

Phylogeny and evolution of SARS-CoV-2 during Delta and Omicron variant waves in India.

SARS-CoV-2 evolution has continued to generate variants, responsible for new pandemic waves locally and globally. Varying disease presentation and severity has been ascribed to inherent variant characteristics and vaccine immunity. This study analyzed genomic data from 305 whole genome sequences from SARS-CoV-2 patients before and through the third wave in India. Delta variant was reported in patients without comorbidity (97%), while Omicron BA.2 was reported in patients with comorbidity (77%). Tissue adaptation studies brought forth higher propensity of Omicron variants to bronchial tissue than lung, contrary to observation in Delta variants from Delhi. Study of codon usage pattern distinguished the prevalent variants, clustering them separately, Omicron BA.2 isolated in February grouped away from December strains, and all BA.2 after December acquired a new mutation S959P in ORF1b (44.3% of BA.2 in the study) indicating ongoing evolution. Loss of critical spike mutations in Omicron BA.2 and gain of immune evasion mutations including G142D, reported in Delta but absent in BA.1, and S371F instead of S371L in BA.1 could explain very brief period of BA.1 in December 2021, followed by complete replacement by BA.2. Higher propensity of Omicron variants to bronchial tissue, probably ensured increased transmission while Omicron BA.2 became the prevalent variant possibly due to evolutionary trade-off. Virus evolution continues to shape the epidemic and its culmination.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2↗
PubMed1976

Isolation of single stranded DNA related to the transcriptional activity of animal cells.

Single stranded DNA (s.s.DNA) comprising 1-2% of the total nuclear DNA was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA3 of embryonic chick cells, labeled for several cell generations with 3H-thymidine. Small quantities of 3H-DNA were annealed with a large excess of unlabeled DNA or polysomal RNA from chick embryos. Hybridization kinetics (monitored by the use of SI nuclease digestion, hydroxyapatite chromatography and thermalfusion), indicated that s.s.DNA belongs to the non repetitious fraction of the cell genome. One third represents DNA sequences engaged in the transcription of messenger RNA's.

Animals↗
PubMed2026

A Cis-Regulatory Duplication in a Hox Hotspot Implicated in Mimetic Convergence in the Bumble Bee Bombus flavifrons.

Several species of North American bumble bees spanning the Pacific Coastal and Rocky Mountain regions converge onto distinct mimetic abdominal colour forms for each region by switching abdominal coloration from black to red. Previous genome-wide association studies (GWAS) of red and black transitions in two mimics (Bombus melanopygus and Bombus vancouverensis) revealed that black forms were generated by independently deleting a portion of the same cis-regulatory region near the Hox gene Abdominal-B (Abd-B). Here, we test the genetic basis of these mimetic colour forms in a third co-mimic, Bombus flavifrons, that has continuous variation in red and black that is shifted posteriorly one segment compared to its co-mimics. Using genome-wide association of red and black forms, we identified a structural variant <&#x2009;50&#x2009;bp away from the deletions in B. melanopygus and B. vancouverensis that was strongly associated with the colour phenotype. Sequencing across mimicry zones and closely related taxa revealed that all red forms of B. flavifrons and monomorphic red close relative Bombus centralis have a 319&#x2009;bp tandem duplication at this locus that has extensive modification to the duplicated copy. Black forms of B. flavifrons from the Cascades also have this duplication but without the modifications, while black forms in the western Rockies mostly lack this duplication, similar to ancestral black forms. This suggests independent mechanisms may regulate the black phenotypes in different populations and that ancestral sorting of variation and/or adaptive introgression generated these phenotypes. This study strengthens support for this Abd-B cis-regulatory region being a hotspot for regulating abdominal coloration in bumble bees, and features the role of regulatory region duplication in creating novel phenotypes.

Animals↗
PubMed1978

Structure of yeast phenylalanine-tRNA genes: an intervening DNA segment within the region coding for the tRNA.

Sixteen bacterial clones containing sequences complementary to yeast PhetRNA were isolated from a collection of hybrid plasmids containing BamHI restriction endonuclease-generated yeast DNA fragments inserted in the plasmid vector pBR315. Ten of these clones contained hybrid plasmids with distinct BamHI fragments. The sequence of the Phe-tRNA structural genes and adjacent regions of three of these clones is reported here. In the region flanking the tRNA gene, the sequence of two of the cloned DNAs is similar; the sequence of the third varies considerably. All three of the tRNA genes are bordered by A,T-rich regions. In particular, near the region coding for the 3' end of the tRNA there is a long sequence of As in the coding strand. This is reminiscent of the region of termination of transcription of the yeast 5S rRNA gene. The sequences coding for the Phe-tRNA contain an additional segment of 18 or 19 base pairs (depending upon the clone) not predicted by the yeast Phe-tRNA sequence. These intervening segments are nearly identical in the three clones and are located within the structural gene, two base pairs from the nucleotides coding for the tRNA anticodon.

Anticodon↗
PubMed2025

The proteogenomic landscape of the human kidney and implications for cardio-kidney-metabolic health.

Nearly one-third of the global population is affected by cardio-kidney-metabolic (CKM) diseases; however, the molecular mechanisms underlying CKM diseases are poorly understood. Here we show that tissue proteomics provide critical insights not captured by tissue gene expression or blood proteomics information by performing whole-genome and RNA sequencing and proteomics analysis of human kidney samples (n&#x2009;=&#x2009;337), and we generated a publicly available database. Via Bayesian co-localization and Mendelian randomization analyses of kidney protein quantitative trait loci and 36 CKM genome-wide association studies, we prioritized 89 proteins for CKM traits. We prioritized relationships that could underlie the interconnectedness of CKM traits and discovered multiple and targetable mechanisms for CKM diseases, including the potential role of kidney angiopoietin-like protein 3 (ANGPTL3) in serum lipid levels and kidney function as well as the role of charged multivesicular body protein 1A in kidney function and hypertension. Notably, we identify pathways with confluence of evidence from genetic loci, tissue gene expression and protein levels for CKM traits. In summary, our large-scale kidney proteomics study uncovers proteins and targetable mechanisms prioritized for CKM diseases.

Humans↗
PubMed1979

Hepatitis B viral DNA molecules have cohesive ends.

Hepatitis B virus DNA made fully double stranded by a virion DNA polymerase reaction could be converted from circular to linear molecules by heating in 10 mM NaCl at 77 degrees C or in 100 mM NaCl at 90 degrees C for 15 min. Heat-generated linear hepatitis B virus DNA was reannealed to circular molecules by incubating in higher salt concentrations. The identity of the molecular forms was established by their electrophoretic mobility and appearance in electron micrographs. Recircularization was blocked by reacting linear molecules with nuclease S1 or avian myeloblastosis virus reverse transcriptase. These results suggest that the heated linear DNA had single-stranded ends with complementary nucleotide sequences. It also suggests that a discontinuity or nick is present in each strand of the circular DNA molecule after the single-stranded region is made double stranded by the virion DNA polymerase reaction. The difference in contour length by electron microscopy of circular and linear molecules spread under aqueous conditions suggested that the discontinuities in the two strands were about 270 base pairs apart. The amount of nucleotide incorporated into the ends of heat-generated linear hepatitis B virus DNA by reverse transcriptase suggested that the single-stranded ends were about 305 bases in length. This fully double-stranded linear DNA was cleaved with EcoRI or HpaI restriction endonuclease. The sum of the two fragments generated by each totaled 3,510 base pairs, 310 base pairs greater than the contour length of circular hepatitis B virus DNA which represents a third estimate of the distance between the discontinuities in the two DNA strands of circular DNA. Restriction endonuclease cleavage also indicated that the ends of heated linear DNA which correspond to the discontinuities in the two strands of the circular DNA are at unique sites in the DNA with respect to the restriction sites.

Base Sequence↗
PubMed1979

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Base Sequence↗
PubMed1975

Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion.

A series of recombinant plasmids was generated in Escherichia coli in which the TEM beta-lactamase translocon (TnA) was inserted into the small plasmid RSF1010. RSF1010 is a 5.5 X 10(6) dalton nonconjugative plasmid which confers resistance to streptomycin and sulfonamide. The recombinant plasmids can be classified into three clearly defined phenotypic groups. Group I is ampicillin-, streptomycin- and sulfonamide-resistant. Group II is ampicillin- and sulfonamide-resistant but has lost streptomycin resistance. Group III is ampicillin-resistant but is sensitive to sulfonamide and shows a simultaneous 30-fold reduction in the minimal inhibitory concentration of streptomycin. It was possible to map the site of insertion of TnA within RSF1010 by electron microscope studies of DNA heteroduplexes formed between RSF1010 and recombinant plasmids. Insertions of TnA occur at, at least, 12 distinct sites in a region corresponding to one-third of the RSF1010 DNA molecule. Those insertions giving rise to particular phenotypes are clustered. Insertions of TnA-like insertion sequences (IS) appear to give rise to strongly polar mutations.

Ampicillin↗
PubMed1979

Sequence analysis of adenovirus DNA: complete nucleotide sequence of the spliced 5' noncoding region of adenovirus 2 hexon messenger RNA.

The complete nucleotide sequence of the 5' noncoding region of the adenovirus 2 hexon messenger RNA has been established by sequence analysis of reverse transcripts. Such transcripts were generated by extension of specific single-stranded DNA primers with reverse transcriptase after hybridization to purified hexon mRNA. The total length of the 5' noncoding region was determined to be 240 nucleotides, of which the spliced tripartite leader sequence contributes 202 nucleotides including the terminal m7G. The sizes of the different segments of the tripartite leader were estimated by comparing the established mRNA sequence with the genomic sequences for the first and third leader segments, and were found to be 42 nucleotides for the first segment, 71 nucleotides for the second and 89 nucleotides for the third. The estimates are ambiguous, however, due to the presence of tandemly repeated sequences at both ends of the intervening sequence between the third leader segment and the body of the hexon mRNA. The sequence of the leader allows the formation of hydrogen-bonded interactions with the 3' end of 18S ribosomal RNA near the capped 5' end and also close to the initiator AUG.

Adenoviruses, Human↗
PubMed1977

Germ line integration of moloney leukemia virus: effect of homozygosity at the m-mulV locus.

Mice genetically transmitting the exogenous Moloney leukemia virus (M-MuLV) have been previously derived. These animals carried one copy of M-MuLV DNA in their germ line and were heterozygous for the M-MuLV locus (Jaenisch, 1976). Experiments were performed to investigate whether homozygosity at the M-MuLV locus would be compatible with normal development. Animals heterozygous for the M-MuLV locus were mated [female (+/-) X male(+/-)] and the genotype of the offspring was analyzed. Molecular hybridization experiments revealed three classes of offspring carrying two copies (++), one copy (+/-) and no (--) M-MuLV-specific DNA sequences, respectively, in their liver DNA. Genetic experiments indicated that males of the first class transmitted the virus to 100% of their offspring, males of the second class to 50% and males of the third class not at all when mated with normal females. These results demonstrated that homozygosity at the M-MuLV locus has no detectable effect on normal development of the animals and that the M-MuLV gene is transmitted from one generation to the next strictly according to Mendelian expectations. Development of M-MuLV-induced leukemia is not influenced by the genotype of these animals--that is, animals carrying two or one copies of M-MuLV in their germ line or animals congenitally infected from the mother developed disease at similar rates.

Animals↗
PubMed2025

Fishing for a reelGene: evaluating gene models with evolution and machine learning.

Assembled genomes and their associated annotations have transformed our study of gene function. However, each new annotated assembly generates new gene models. Inconsistencies between annotations likely arise from biological and technical causes, including pseudogene misclassification, transposon activity, and intron retention from sequencing of unspliced transcripts. To evaluate gene model predictions, we developed reelGene, a pipeline of machine learning models focused on (1) transcription boundaries, (2) mRNA integrity, and (3) protein structure. The first two models leverage sequence characteristics and evolutionary conservation across related taxa to learn the grammar of conserved transcription boundaries and mRNA sequences, while the third uses the conserved evolutionary grammar of protein sequences to predict whether a gene can produce a protein. Evaluating 1.8 million transcript models in Zea mays ssp. mays (maize), reelGene classified 28% as incorrectly annotated or non-functional. We find that reelGene classifies 92.2% of genes in the maize proteome and 99.2% of genes within the maize classical gene list as functional. reelGene also provides a way to further investigate genome biology- for instance, reelGene indicates that 10.3% of dispensable genes in B73 are functional, and within retained duplicate genes, reelGene identifies a 30% bias toward the retention of the M1 subgenome when one copy is functional and the other is non-functional. As an annotation-evaluating tool, reelGene is directly applicable to species of the Andropogoneae tribe, including other important crops like sorghum and miscanthus. As a community resource, reelGene has been integrated onto MaizeGDB both as a browser track and as an individual Shiny App, allowing researchers to evaluate gene model accuracy and further investigate genome biology.

Machine Learning↗
PubMed1978

Immunoglobulin light chain mRNA is processed from large nuclear RNA.

Recombinant DNA probes, produced by the molecular cloning of immunoglobulin kappa light chain mRNA, have been used to analyze heterogenous nuclear RNA for presumptive precursors to cytoplasmic kappa light chain mRNA, Three discrete classes of nuclear RNA containing kappa mRNA sequences were detected after pulse-labeling of immunoglobulin-producing P3 myeloma cells. Two of these were substantially larger than kappa mRNA (approximately 10 and 4 times larger); the third was similar in size to kappa mRNA. Beginning with the largest, the sequential appearance of these three classes of nuclear RNA preceded the first appearance of newly synthesized kappa light chain mRNA in the cytoplasm. The results presented here suggest that immunoglobulin kappa light chain mRNA is generated by the stepwise cleavage and processing of a large nuclear RNA transcript.

Cell Line↗
PubMed2026

Arabidopsis TITAN-LIKE is required for U12-type intron splicing, especially of AT-AC subtypes.

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes >99% of all introns, whereas the U12-dependent spliceosome acts on only ~0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human centrosomal AT-AC splicing factor (CENATAC) is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN-LIKE (TTL), but its function in splicing remains unknown. Here, we generated ttl mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL's function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than in a drol1 (defective repression of the OLE3:LUC1) mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

Arabidopsis↗
PubMed2026

Genome characterization of two novel mitoviruses and a negative-sense single-stranded RNA mycovirus from the phytopathogenic fungus Clarireedia jacksonii.

Clarireedia jacksonii is a phytopathogenic fungus responsible for dollar spot disease in turfgrass worldwide. In this study, we characterized the complete genome sequences of three novel mycoviruses isolated from C. jacksonii isolate MBCT-836 using next-generation sequencing and the fragmented and primer-ligated dsRNA sequencing (FLDS) method. Two of these viruses, designated Clarireedia jacksonii mitovirus 1 (CjMV1) and Clarireedia jacksonii mitovirus 2 (CjMV2), possess positive-sense single-stranded RNA genomes of 2,575 bp and 2,856 bp, respectively. Both viruses contain a single open reading frame that utilizes the mitochondrial genetic code and encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis placed CjMV1 and CjMV2 within the genera Unuamitovirus and Duamitovirus, respectively, in the family Mitoviridae. The third virus, Clarireedia jacksonii negative-stranded RNA virus 1 (CjNSV1), features a bisegmented negative-sense RNA genome consisting of a large segment (7,961 nt) encoding an RdRp with a conserved Bunya_RdRp domain, and a small segment (1,444 nt) encoding a protein showing homology to bunyavirus nucleocapsid proteins. Phylogenetic analysis revealed that CjNSV1 clusters with members of the proposed family Sclerobunyaviridae within the order Bunyavirales. To our knowledge, this study provides the first report of complete genome sequences of mycoviruses infecting C. jacksonii, expanding our understanding of the mycovirosphere in economically significant turfgrass pathogens.

Genome, Viral↗
PubMed1979

Application of linear system analysis to the horizontal vestibulo-ocular reflex of the alert rhesus monkey using pseudorandom binary sequence and single frequency sinusoidal stimulation.

Horizontal eye movements of the alert rhesus monkey resulting from both pseudorandom binary sequence (PRBS) and single frequency sinusoidal rotational stimulation were analyzed using a PDP 11/40 computer in order to generate gain, phase, and coherence estimates at discrete frequencies between 0.008 and 1.28 Hz. A computer simulation of vestibular induced eye movements was used to validate our analysis procedures and to determine the effects of digital noise. Frequency domain transfer functions derived from gain and phase estimates revealed that the responses to PRBS stimulation and to single frequency sinusoids were not appreciably different. PRBS testing was accomplished in approximately one third the time required for sinusoidal testing and yielded highly reproducible data. We conclude that PRBS stimulation is a reliable and efficient method for assessing linear system parameters of the horizontal vestibulo-ocular reflex. PRBS testing may be particularly advantageous in studies of vestibulo-oculomotor plasticity in which rapid assessment of alterations in system dynamics is essential.

Animals↗