Wild-type Chromosome Research Articles

ORFless, intronless, and mutant transcription units in the mouse t complex responder (Tcr) locus.

Mouse t haplotypes exhibit transmission ratio distortion (TRD), whereby heterozygous males (+/t) transmit the t chromosome to nearly all offspring. TRD is mediated by the t complex responder locus (Tcr), whose transmission is elevated above Mendelian levels by additive contribution of several t haplotype-encoded quantitative trait loci (QTLs) called t complex distorters (Tcd1-Tcd5). The entire genetically defined Tcr interval has been cloned and consists of under 200 kb. This interval is one of three large duplication units in t haplotypes that retain high levels of similarity. The cis-active nature of Tcr raises the possibility that it is not a protein-encoding gene, but another anomaly such as a structural anomaly of chromatin. To further investigate the Tcr-critical interval, a 30-kb region upstream of the Tcp10b(t) gene (a testis-expressed former candidate for Tcr) was sequenced, along with the duplicated paralogous region associated with Tcp10c(t), which lies immediately adjacent but outside the Tcr interval. Several genes or transcriptional units were identified, including the 3' end of ribosomal s6 kinase (Rsk3); two apparently intronless and ORF-less genes; and Gpr31, an intronless, putative G-protein coupled receptor. While the 30-kb regions were 98% identical, the Gpr31 paralog from the Tcr-critical region (Gpr31b(t)) contained an in-frame 210-bp deletion that disrupted two of the seven predicted transmembrane domains. Furthermore, an intronless and ORFless gene from this interval, Trex1b(t), contained a 4-bp deletion that distinguished it from all other homologs in t haplotypes and wild-type chromosomes. Although it is unknown whether any of these genes are involved in TRD, their discovery raises new possibilities regarding the nature of Tcr, including a model whereby it might function as an RNA rather than a protein or chromatin anomaly.

Engineering a mouse balancer chromosome

Balancer chromosomes are genetic reagents that are used in Drosophila melanogaster for stock maintenance and mutagenesis screens. Despite their utility, balancer chromosomes are rarely used in mice because they are difficult to generate using conventional methods. Here we describe the engineering of a mouse balancer chromosome with the Cre-loxP recombination system. The chromosome features a 24-centiMorgan (cM) inversion between Trp53 (also known as p53) and Wnt3 on mouse chromosome 11 that is recessive lethal and dominantly marked with a K14-Agouti transgene. When allelic to a wild-type chromosome, the inversion suppresses crossing over in the inversion interval, accompanied by elevated recombination in the flanking regions. The inversion functions as a balancer chromosome because it can be used to maintain a lethal mutation in the inversion interval as a self-sustaining trans-heterozygous stock. This strategy can be used to generate similar genetic reagents throughout the mouse genome. Engineering of visibly marked inversions and deficiencies is an important step toward functional analyses of the mouse genome and will facilitate large-scale mutagenesis programs.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

Estimating loss of the wild-type p53 gene by in situ hybridization of fine-needle aspirates from breast carcinomas.

TP53 mutations have been found in 16-64% of breast carcinomas. The aim of our study was to investigate loss of the wild-type TP53 gene by in situ hybridization (ISH) of fine-needle aspirates (FNAC) from breast carcinomas. The material consisted of FNAC from 33 breast carcinomas, with histologic specimens from 19 of the cases. Routine diagnostic smears were used for cytologic grading. ISH of the wild-type TP53 gene and chromosome 17 was performed on air-dried smears. Hybridization signals were counted in at least 100 nuclei, and the percentage for each signal number was calculated. FNAC from four fibroadenomas as well as cell preparations from five lymphocyte cultures were used as normal/benign controls. Cutoff for defining loss of p53 gene signals was set at 20% of cells with zero and one gene signal only. Concomitant p53 protein expression was determined on 20 histologic sections and eight additionally available air-dried smears. Loss of wild-type p53 gene was found in 20 carcinomas (60.6%). The rate of signal loss varied from 0.4% to 75.3% of the cells. All tumors with aneusomy of chromosome 17 revealed loss of p53 gene signals, as did 42% of the disome cases. Loss of wild-type p53 gene was present in 10 of 16 grade 1 cancers (62.5%), eight of 13 grade 2 tumors (61.5%), and two of four grade 3 cases. Signal loss did not correlate with p53 protein expression. In conclusion, subpopulations with loss of the wild-type p53 gene are a common finding in breast carcinomas; they are detected in more than 60% of the tumors, including grade 1 cancers.

Myxococcus xanthus sasN encodes a regulator that prevents developmental gene expression during growth.

Myxococcus xanthus multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (sasB5, -14, -15, -16, and -17) can bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. These mutants express 4521 at high levels during growth and development in an asgB background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the sasB locus cloned from the wild-type chromosome restored the SasB+ phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed sasN-null mutation and Omega4521 Tn5lac in an otherwise wild-type background expressed 4521 at a high level during growth and development. A similar sasN-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body development and functions as a critical regulator that prevents 4521 expression during growth.

In vitro loss of heterozygosity targets the PTEN/MMAC1 gene in melanoma.

Gross genetic lesions of chromosome 10 occur in 30-50% of sporadic human melanomas. To test the functional significance of this observation, we have developed an in vitro loss of heterozygosity approach in which a wild-type chromosome 10 was transferred into melanoma cells, where there was selection for its breakage and regional deletion to relieve its growth suppressive effects. The overlap of these events was at band 10q23, the site of the recently isolated PTEN/MMAC1 tumor suppressor gene, suggesting it as a potential target. Although the gene was expressed in the parental cells, both of its chromosomal alleles contained truncating mutations. In vitro loss of heterozygosity resulted in loss of the chromosomally introduced wild-type PTEN/MMAC1, and ectopic expression of the gene caused cell growth suppression. Thus, this approach identified PTEN/MMAC1 as a target in malignant melanoma and may provide an alternative means to localizing tumor suppressor genes.

Genetic instability of the Streptomyces chromosome.

The Streptomyces wild-type chromosome is linear in all examples studied. The ends of the chromosome or telomeres consist of terminal inverted repeats of various sizes with proteins covalently bound to their 5' ends. The chromosome is very unstable and undergoes very large deletions spontaneously at rates higher than 0.1% of spores. Frequently, the telomeres are included in the deletions. Loss of both telomeres leads to circularization of the chromosome. The wild-type chromosome can also be circularized artificially by targeted recombination. Spontaneously or artificially circularized chromosomes are even more unstable than the linear ones. High-copy-number tandem amplifications of specific chromosomal regions are frequently associated with the deletions. RecA seems to be involved in the amplification mechanism and control of genetic instability.

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced M r of 40 700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M· NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.

The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes.

The virulence of the human pathogen Shigella flexneri is dependent on both chromosome- and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which encodes the tRNA N6-isopentyladenosine (i6A37) synthetase and is involved in the first step of the synthesis of the modified nucleoside 2-methylthio-N6-isopentenyladenosine (ms2i6A), was transferred to the chromosome of S. flexneri 2a by phage P1 transduction. In the wild-type bacterium, ms2i6A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The miaA::Km Sma mutant of S. flexneri accordingly lacked ms2i6A37 in its tRNA. In addition, the mutant strains showed reduced expression of the virulence-related genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold-reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of the wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms2i6A in the tRNA of the miaA mutants. In the miaA mutant, the level of the virulence-associated protein VirF was reduced 10-fold compared with the wild type. However, the levels of virF mRNA were identical in the mutant and in the wild type. These findings suggest that a posttranscriptional mechanism influenced by the presence of the modified nucleoside ms2i6A in the tRNA is involved in the expression of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coli was further generalized.

The sequence of palF, an environmental pH response gene in Aspergillus nidulans

To molecularly characterize the influence of external pH in the secretion of enzymes by filamentous fungi, we have cloned and sequenced the palF gene of Aspergillus nidulans ( An). An An wild-type (wt) chromosome VII-specific cosmid library was used to transform a palF15 mutant strain. Selection for complementation was done on medium containing β-glycerolphosphate as the sole P i source. Two cosmids were identified (W2G08 and W4G12) and further subcloning of one cosmid (W2G08) defined a 5-kb PstI genomic fragment, which fully complements the palF15 mutation. An internal fragment from the genomic clone recognized a single message of approx. 3.5 kb on Northern blot. cDNA clones were obtained from a λgt10 cDNA library and sequenced, showing a nucleotide (nt) sequence of 3311 base pairs (bp), with a 828 bp long 5′-untranslated region ( UTR). The major open reading frame (ORF) identified in the sequence codes for a putative 775 amino acid (aa) protein, which shares some similarity with two putative ORF products of Saccharomyces cerevisiae ( Sc).

The yeast Red1 protein localizes to the cores of meiotic chromosomes.

Mutants in the meiosis-specific RED1 gene of S. cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC. Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes. Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs. Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC. Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination. The localization of Red1 has been compared with two other meiosis-specific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes. Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene. Double labeling with anti-Hop1 and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function. These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact. There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.

Genetic variation for total fitness in Drosophila melanogaster.

We measured the heterozygous effects on net fitness of a sample of 12 wild-type third chromosomes in D. melanogaster. Effects on fitness were assessed by competing the wild-type chromosomes against balancer chromosomes, to prevent the production of recombinants. The measurements were carried out in the population cage environment in which the life history had been evolving, in an undisturbed population with overlapping generations, and replicated measurements were made on each chromosome to control for confounding effects such as mutation accumulation. We found significant variation among the wild type chromosomes in their additive genetic effect on net fitness. The system provides an opportunity to obtain an accurate estimate of the distribution of heterozygous effects on net fitness, the contribution of different fitness components including male mating success, and the role of intra-chromosomal epistasis in fitness variation.

Beaded of Goldschmidt, an antimorphic allele of Serrate, encodes a protein lacking transmembrane and intracellular domains.

Serrate (Ser) is an essential gene in Drosophila melanogaster best known for the Ser dominant (SerD) allele and its effects on wing development. Animals heterozygous or homozygous for SerD are viable and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt (BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus with a single 5' Ser-homologous region and two distinct 3' regions. Meiotic recombination between BdG and a wild-type Ser chromosome demonstrated that only one DNA lesion, caused by the insertion of a transposable roo element into the coding regions of the Ser transcript, appears capable of generating BdG phenotypes. Due to the roo insertion, the protein product is predicted to be prematurely truncated and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains found within the normal SERRATE (SER) protein. The loss of these protein domains apparently contributes to the antimorphic nature of this mutation.

Chromosomal localization of the neurological mouse mutations tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr).

We have refined the map positions and identified molecular markers for three neurological mutations in the mouse, tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr). These mutations were localized using simple sequence length polymorphisms between the mouse strain on which the mutation arose and the inbred strain onto which the mutation was bred. This approach to mutation mapping is generalizable to any mutant that has been backcrossed for several generations. The tg mutation was localized to the 1.1 cM region of chromosome 8 distal to simple sequence repeat (SSR) D8Mit103 and proximal to SSRs D8Mit79, D8Mit105, and D8Mit283. The pcd locus was mapped to the 5 cM interval of chromosome 13 between SSRs D13Mit139 and D13Mit67, and the nr locus was mapped between SSRs D8Mit155 and D8Mit18, a 5.6 cM region of chromosome 8. For each mutation, several SSRs distinguishing mutant from wild type chromosomes were identified within these regions. The definition of molecular markers distinguishing mutant from wild type alleles makes possible for the first time identification of tg, pcd, and nr mutants prior to behavioral manifestation of the mutant genotype. Thus, developmental studies of these mutants designed to describe or dissect the biochemical basis of the induction of the mutant phenotype are now feasible.

The Triplo-lethal locus of Drosophila: reexamination of mutants and discovery of a second-site suppressor.

In the genome of Drosophila melanogaster there is a single locus, Triplo-lethal (Tpl), that causes lethality when present in either one or three copies in an otherwise diploid animal. Previous attempts to mutagenize Tpl produced alleles that were viable over a chromosome bearing a duplication of Tpl, but were not lethal in combination with a wild-type chromosome, as deficiencies for Tpl are. These mutations were interpreted as hypomorphic alleles of Tpl. In this work, we show that these alleles are not mutations at Tpl; rather, they are dominant mutations in a tightly linked, but cytologically distant, locus that we have named Suppressor-of-Tpl (Sul(Tpl)). Su(Tpl) mutations suppress the lethality associated with three copies of the Triplo-lethal locus and are recessive lethal. We have mapped Su(Tpl) to the approximate map position 3-46.5, within the cytological region 76B-76D.

Physical mapping of the Tec and Gabrb1 loci reveals that the Wsh mutation on mouse chromosome 5 is associated with an inversion.

In the mouse, mutations in the c-Kit proto-oncogene, a member of the receptor tyrosine kinase (RTK) gene family, have pleiotropic effects on hematopoiesis, pigmentation and fertility (dominant spotting, W). However, in the Wsh allele the defect is confined to abnormal pigmentation caused by the disruption of 5' regulatory sequences of Kit leaving an intact structural gene. In this report, the previously published physical map around the Pdgfra-Kit-Flk1 RTK loci is extended by mapping the loci encoding the GABAA (gamma-aminobutyric acid) receptor subunit beta 1, Gabrb1 and a cytoplasmic kinase (Tec) 3 Mb proximal to Kit. PFGE analysis of the wild-type (C57BL/6J) chromosome demonstrates the following gene order: cen-Gabrb1-Tec-Pdgfra-Kit, whereas the analysis of Wsh/Wsh DNA is consistent with the order: cen-Gabrb1-Pdgfra-Tec-Kit. This altered physical map can be explained by an inversion on the Wsh chromosome located proximally to the Kit locus and spanning the 2.8 Mb Pdgfra-Tec chromosomal segment. This high resolution physical mapping study identifies large DNA fragments that span the two inversion breakpoints and potentially carry Kit upstream regulatory elements involved in the control of Kit expression during embryonic development.

Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae.

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface. In this study, the tbpA and tbpB genes of H. influenzae, encoding the transferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned and sequenced. Alignments of the H. influenzae Tbp1 and Tbp2 protein sequences with those of related proteins from heterologous species were analyzed. On the basis of similarities between these and previously characterized proteins, Tbp1 appears to be a member of the TonB-dependent family of outer membrane proteins while Tbp2 is lipid modified by signal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamycin resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome. Binding assays with the mutants showed that a significant reduction in transferrin-binding ability resulted from the loss of either of the Tbps and a complete loss of binding was evident when neither protein was expressed. Loss of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1+ Tbp2- mutant was able to grow only at high transferrin concentrations.

44] DNA replication of bacteriophage T4 in Vivo

This chapter discusses various methods used to investigate bacteriophage T4 DNA replication in vivo. The T4 genome is large for a bacterial virus. It comprises about 168,800 base pairs. It encodes most of the T4 DNA replication and recombination proteins (except for host RNA polymerase) and enzymes that degrade the host DNA. Two properties of the T4 chromosomes are important when considering DNA replication. First, the ends of individual chromosomes are randomly permuted over the circular map. Second, the glucosylation and hydroxymethylation of the cytosine residues protect T4 DNA from the T4 enzymes that degrade the host DNA, as well as from most of the known restriction enzymes. Origin initiation in most wild-type T4 chromosomes is bidirectional. In primase- or topoisomerase-deficient mutants, initiation from oriA or oriF is unidirectional in the direction of transcription, as expected if transcripts prime leading strand synthesis in one direction, but primase is required to prime Okazaki pieces so that the first Okazaki piece becomes the leading strand in the direction opposite to that of transcription. Probably, topoisomerase has to resolve the ensuing entanglement of DNA strands. The T4 replication origins are mainly characterized by hybridizations of in vivo labeled nascent T4 DNA, isolated early after infection, to Southern blots of cloned T4 fragments or restriction digests of unmodified T4 DNA and by electron microscopy.

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using this procedure, clones of the restriction-deficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orf1 and orf2) on the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterium lilium ATCC 15990.

Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number.

Drug resistance is a significant impediment to the therapy of African sleeping sickness in humans. To evaluate molecular mechanisms that contribute to drug resistance in African trypanosomes, a clonal strain of Trypanosoma brucei gambiense, MPA10, was selected in medium containing mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. IMPDH activity in MPA10 cells was approximately 6-fold higher than that of wild type parental cells, although the enzymes in both strains were equally sensitive to inhibition by MPA. To evaluate the mechanism of IMPDH overexpression in MPA10 cells, the gene encoding IMPDH (impdh) was isolated from a T.b. brucei library by cross-hybridization to the Leishmania donovani impdh. Sequence analysis indicated that the T. brucei IMPDH was 76% identical with the L. donovani IMPDH. The T. brucei impdh hybridized to a 2.7-kb transcript that was expressed at approximately 10-fold greater levels in the MPA10 cells, and this impdh overexpression could be ascribed to an approximately 10-fold amplification of the impdh copy number. Pulsed field gel electrophoresis revealed that the extra impdh copies in MPA10 cells were localized to an approximately 6.0-Mb chromosome that comigrated with the wild type chromosome encompassing impdh. The amplification of impdh could be ascribed to an increased copy number of this 6.0-Mb chromosome, and a predicted augmented DNA content in MPA10 cells was confirmed by flow cytometry. This is the first demonstration that DNA amplification can serve as a molecular mechanism by which T. brucei become resistant to cytotoxic drugs, and the amplification of the 6.0-Mb chromosome represents a novel mechanism of drug resistance in parasitic protozoa.