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Genetic bias in immune responses to a cassette shared by different microorganisms in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with HLA-DRbeta1 alleles which contain the QKRAA amino acid sequence in their third hypervariable region(s). The QKRAA sequence is also expressed by several human pathogens. We have shown previously that an Escherichia coli peptide encompassing QKRAA is a target of immune responses in RA patients. Here we address two questions: first, whether QKRAA may function as an "immunological cassette" with similar, RA-associated, immunogenic properties when expressed by other common human pathogens; and second, what is the influence of genetic background in the generation of these responses. We find that early RA patients have enhanced humoral and cellular immune responses to Epstein-Barr virus and Brucella ovis and Lactobacillus lactis antigens which contain the QKRAA sequence. These results suggest that the QKRAA sequence is an antigenic epitope on several different microbial proteins, and that RA patients recognize the immunological cassette on different backgrounds. ANOVA of immune responses to "shared epitope" antigens in monozygotic twin couples shows that, despite significantly elevated responses in affected individuals, a similarity between pairs is retained, thus suggesting a role played either by hereditary or shared environmental factors in the genesis or maintenance of these responses.

Antigen Presentation↗

Identification of differentially expressed genes in human prostate cancer using subtraction and microarray.

We have identified human prostate cancer- and tissue-specific genes using cDNA library subtraction in conjunction with high throughput microarray screening. Subtracted cDNA libraries of prostate tumors and normal prostate tissue were generated. Characterization of subtracted libraries showed enrichment of both cancer- and tissue-specific genes. Highly redundant clones were eliminated by colony hybridization. The remaining clones were selected for microarray to determine gene expression levels in a variety of tumor and normal tissues. Clones showing overexpression in prostate tumors and/or normal prostate tissues were selected and sequenced. Here we report the identification of two genes, P503S and P504S, from subtracted libraries and a third gene, P510S, by subtraction followed by microarray screening. Their expression profiles were further confirmed by Northern blot, real-time PCR (TaqMan), and immunohistochemistry to be overexpressed in prostate tissues and/or prostate tumors. Full-length cDNA sequences were cloned, and their subcellular locations were predicted by a bioinformatic algorithm, PSORT, to be plasma membrane proteins. The genes identified through these approaches are potential candidates for cancer diagnosis and therapy.

Blotting, Northern↗

[Regulation of G proteins by receptors].

Receptors located on the cell surface are responsible for recognition of extracellular first messengers. The largest number of receptors belongs to a G protein-coupled receptor superfamily. They showed common structural features characterized by seven transmembrane regions and three connecting extracellular and intracellular loops. Stimulation of a receptor by an agonist activates the coupled G protein. From in vitro mutagenesis analyses of beta 2-adrenergic, muscarinic acetylcholine and alpha 1-adrenergic receptors, it was shown that the intracellular third loop and carboxyl terminus of the receptor molecule are involved in coupling with a G protein. One intriguing observation was that mutation at a single site of the intracellular third loop could induce the active state of receptors without agonist stimulation. Receptor heterogeneity generated from distinct genes or alternative splicing can be seen at the intracellular third loop and carboxyl terminus that is assumed to play an important role of coupling with G proteins. There are several examples that receptor isoforms arising from alternative splicing have their own counterparts of G proteins.

Amino Acid Sequence↗

Two distinct human endothelin B receptors generated by alternative splicing from a single gene.

A novel variant of endothelin B receptor (ETB) has been found in human brain, placenta, lung, and heart by reverse transcriptase polymerase chain reaction. This variant ETB1 has an additional 30 nucleotide sequence with splice sites at both ends. This results in a 10 amino acid increase in the length of the second cytoplasmic domain of ETB. Polymerase chain reaction on genomic DNA indicates that this sequence is part of the 134 bp intron which separates the second and third exons and is contiguous with the third exon of the ETB gene. Southern blot analysis of chromosomal DNA and genomic PCR results indicate that ETB1 arises by alternative RNA splicing of the single copy ETB gene. The insert sequence in ETB1 gene is absent in bovine, rat, and porcine DNA, and is unique to human DNA. Both ETB and ETB1 have been expressed in heterologous systems to examine their ligand binding and functional properties. Reverse transcriptase polymerase chain reaction of RNA from ETB1 expressing cells indicates that the additional sequence is stably expressed.

Alternative Splicing↗

Contingency locus in ctsR of Listeria monocytogenes Scott A: a strategy for occurrence of abundant piezotolerant isolates within clonal populations.

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10(-5). Subsequent sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n = 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within L. monocytogenes cultures, thus playing an important role in survival.

Amino Acid Sequence↗

Altered repair of targeted psoralen photoadducts in the context of an oligonucleotide-mediated triple helix.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplex-directed lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.

Animals↗

Tissue-specific and ubiquitous promoters direct the expression of alternatively spliced transcripts from the calcitonin receptor gene.

The gene encoding the murine calcitonin receptor (mCTR) was isolated, and the exon/intron structure was determined. Analysis of transcripts revealed novel cDNA sequences, new alternative exon splicing in the 5'-untranslated region, and three putative promoters (P1, P2, and P3). The longest transcription unit is greater than 67 kilobase pairs, and the location of introns within the coding region of the mCTR gene (exons E3-E14) are identical to those of the porcine and human CTR genes. We have identified novel cDNA sequences that form three new exons as well as others that add 512 base pairs to the 5' side of the previously published cDNA, thereby extending exon E1 to 682 base pairs. Two of these novel exons are upstream of exon E2 and form a tripartite exon E2 (E2a, E2b, and E2c) in which E2a is utilized by promoter P2 with variable splicing of E2b. The third new exon (E3b') lies between E3a and E3b and is utilized by promoter P3. Analysis of mCTR mRNAs has revealed that the three alternative promoters give rise to at least seven mCTR isoforms in the 5' region of the gene and generate 5'-untranslated regions of very different lengths. Analysis by reverse transcription-polymerase chain reaction shows that promoters P1 and P2 are utilized in osteoclasts, brain, and kidney, whereas promoter P3 appears to be osteoclast-specific. Using transiently transfected reporter constructs, promoter P2 has activity in both a murine kidney cell line (MDCT209) and a chicken osteoclast-like cell line (HD-11EM), whereas promoter P3 is active only in the osteoclast-like cell line. These transfection data confirm the osteoclast specificity of promoter P3 and provide the first evidence that the CTR gene is regulated in a tissue-specific manner by alternative promoter utilization.

Alternative Splicing↗

Identification of a human cDNA encoding a novel Bcl-x isoform.

Alternative splicing has been shown to generate two isoforms of the apoptosis regulator bcl-x, Bcl-xL and Bcl-xS, in humans. Here we describe the identification and characterization of a third splice variant of the human bcl-x gene. It differs from previously described bcl-x transcripts in two respects: (1) a novel facultative intron is spliced out at the 5' untranslated region and (2) the open reading frame arises from a continuous genomic sequence extending over the splice donor sites utilized by the bcl-xL and bcl-xS transcripts. Since the resulting molecule has an organisation homologous to mouse and rat Bcl-x beta we suggest calling this novel protein human Bcl-x beta. Northern blot analysis revealed that bcl-x beta mRNA is expressed in numerous cell lines. Like Bcl-xL, h-Bcl-x binds to the pro-apoptotic protein Bax, suggesting a functional activity in vivo.

Amino Acid Sequence↗

White matter and lesion T1 relaxation times increase in parallel and correlate with disability in multiple sclerosis.

Previous studies have established the clinical relevance of hypointense lesions ("black holes") on T1-weighted MRI as a surrogate marker for pathological change [36]. In contrast to measuring the volume of "black holes", the direct measurement of T1 values allows an objective assessment of the changes contributing to hypointensity both in the focal lesions and in the normal appearing white matter (NAWM). The aims of this study were first, to determine the relationship between T1 values in the NAWM and in discrete lesions, second, to test the relationship between white matter T1 changes and measures of disability and third, to determine whether pathology leading to T1 change occurred in thalamic grey matter of patients with multiple sclerosis. 24 patients with clinically definite multiple sclerosis (13 with relapsing-remitting multiple sclerosis and 11 with secondary progressive multiple sclerosis) and 11 controls participated. White matter T1 histograms and mean T1 values for the thalamus were generated from whole brain T1 relaxation time maps measured using a novel echo-planar imaging based MRI sequence at 3Tesla. Tissue segmentation based on T2- and T1-weighted images allowed independent study of changes in lesions and NAWM. White matter T1 histograms from the patient group showed a reduced peak height and a shift towards higher T1 values (p = 0.028) relative to controls. The mean thalamic T1 was greater for secondary progressive patients than for healthy controls (p = 0.03). Mean white matter T1 values correlated significantly with disability (r = 0.48, p = 0.02). The mean T1 value in the T1-hypointense lesions correlated strongly with the mean T1 value in the NAWM (r = 0.80, p < 0.001). No significant relationship was found between mean white matter T1 value and cerebral volume (r = -0.23, p = 0.31). The T1 measurements extend previous observations suggesting that changes in the NAWM occur in parallel with pathology in lesions of MS. T1 measurements of either the total or NAWM therefore may provide a potentially observer- and scanner- independent marker of pathology relevant to disability in MS.

Adult↗

Rapid isolation of CA microsatellites from the tilapia genome.

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.

Animals↗

The human p167 gene encodes a unique structural protein that contains centrosomin A homology and associates with a multicomponent complex.

The characterization of novel cytoplasmic, structural, and enzymatic proteins has been enhanced by a panel of monoclonal antibodies specific for protein substrates of transforming and nontransforming c-Src mutants. These protein substrates have included the focal adhesion kinase (FAK), cortactin, AFAP-110, p120CAS, and p130CAS. The monoclonal antibody 4G8 was generated as part of this panel of antibodies and was used to isolate the human gene for a 167-kD polypeptide. The cDNA sequence is 5,238 nucleotides in length with a predicted open reading frame consisting of 1,382 amino acids. The polypeptide is largely hydrophilic and highly charged. The central region of p167 has 88% identity with the entire 278-amino-acid encoded sequence of the murine centrosomin A gene. The carboxyl third of p167 contains a unique cluster of 10 amino acid repeats with the consensus sequence (A/M)DDDRGPRRG. The p167 protein was found primarily in the cytoplasm of lymphocytes and is part of a multicomponent protein complex with prominent members of 167, 120, 64, 45, 40, 38, and 25 kD. Finally, we illustrate the conservation of p167 and its associated complex, and demonstrate its expression in different human tissues and cell types. The data suggest that p167 is novel and has an important cellular function as a cytoplasmic structural protein.

Amino Acid Sequence↗

Characterization of a soluble form of human CD4. Peptide analyses confirm the expected amino acid sequence, identify glycosylation sites and demonstrate the presence of three disulfide bonds.

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class II major histocompatibility (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells. This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P.J. et al. (1985) Cell 42, 93-104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345. The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-beta-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no O-linked oligosaccharides.

Amino Acid Sequence↗

Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection.

A method for quantitating human immunodeficiency virus type 1 plasma viremia may be useful in monitoring disease progression and the responsiveness of patients to a therapeutic regimen or vaccine. A quantitative assay for viral RNA in plasma or sera that differs in several aspects from those reported previously was developed. First, whereas conventional reverse transcriptase-PCR assays involve a two-step process and use two enzymes, the method described uses a single enzyme, rTth DNA polymerase, for both reverse transcription and PCR. The reactions are carried out in a single tube and with a single buffer solution with uninterrupted thermal cycling. Second, uracil-N-glycosylase and dUTP are incorporated into the reaction mixtures to ensure that any carryover of DNA from previous amplifications will not compromise quantitation. Third, a quantitation standard is incorporated into each reaction mixture so that differences in amplification efficiency caused by sample interferents, variability in reaction conditions, or thermal cycling can be normalized. To ensure comparable amplification efficiency, the quantitation standard has the same primer-binding regions as the human immunodeficiency virus type 1 target and generates an amplified product of the same size and base composition. The probe-binding region was replaced with a sequence that can be detected separately. Fourth, a colorimetric detection format was modified to provide at least a four-log-unit dynamic range. The quantitative assay requires only a single amplification of the sample and can be completed in less than 8 h. The procedure was used on archival samples to demonstrate the viremic spike in acute infection and the suppressed levels of circulating virus following seroconversion.

Acute Disease↗

Color transitions in coral's fluorescent proteins by site-directed mutagenesis.

BACKGROUND: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters. RESULTS: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. CONCLUSIONS: Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.

Amino Acid Sequence↗

Production of transgenic mice.

A "transgenic" mouse is identified by the integration of a foreign DNA into its genome. Such animals serve as experimental systems for the study of gene expression and are often generated as models for human diseases. Direct microinjection of DNA into the male pronucleus of a mouse zygote has been the method most extensively used in the production of transgenic mice. Our method is subdivided into three sections: First, Preinjection, where the animals, the donor eggs, and the injection tools are presented. Second, Injection, where the egg handling and micromanipulation is described. Third, Postinjection, where the surgical transfer into pseudopregnant females completes the procedural sequence. Additional sections are provided to include the materials we use and to offer a series of technical tips that cover various aspects of the transgenic process.

Animals↗

On a domain structure of colicin E1. A COOH-terminal peptide fragment active in membrane depolarization.

A prolonged digestion of colicin E1 with dilute trypsin results in the generation of a trypsin-resistant peptide fragment of the colicin which is approximately one-third of the molecule. The amino acid composition of the fragment, Mr = 20,000, is more nonpolar in nature than the colicin, with the major change in composition being the reduction of the arginine content from 25 residues in the Mr = 56,000 colicin molecule to approximately 1 residue in the fragment. The NH2-terminal amino acid sequence of the tryptic fragment shows no similarity to that of colicin E1. Alignment of this sequence with the complete amino acid sequence of the colicin E1 molecule obtained by others, shows that the fragment occupies all, or almost all, of the COOH-terminal section of the molecule. The fragment behaved similarly to colicin E1, in being able to dissipate a potassium diffusion potential in unilamellar membrane vesicles made of dimyristoylphosphatidylcholine. The fragment was able to dissipate the diffusion potential above and below the temperature region (Tm = 23.5-24 degrees C) of the lipid phase transition in these vesicles, consistent with a channel-like function. The fragment did not show measurable binding to colicin receptor sites on the cell surface, but was much more efficient than colicin E1 in its ability to inhibit proline transport by inner membrane vesicles of Escherichia coli. These data imply that a membrane channel-forming function of the molecule is located in this 20,000 molecular weight region at the COOH-terminal end of the colicin molecule.

Amino Acid Sequence↗

Sorsby's fundus dystrophy in a family with a Ser-181-CVS mutation in the TIMP-3 gene: poor outcome after laser photocoagulation.

PURPOSE: Mutations in the TIMP-3 (tissue inhibitor of metalloproteinase-3) gene can cause Sorsby's fundus dystrophy (SFD) and lead to choroidal neovascular membrane (CNV) formation. We studied a large American family of Irish Protestant descent with CNV inherited as an autosomal dominant trait, to determine the phenoptype and to learn whether a mutation was present in TIMP-3. METHODS: Twelve members of 5 generations were evaluated clinically, with psychophysical and electroretinographic testing, and by fluorescein angiography. Blood samples for DNA extraction were obtained from 21 affected and unaffected family members and from 1 unrelated spouse. DNA sequence was determined, and affected individuals showed a Ser-181-Cys mutation in TIMP-3 exon 5. RESULTS: Observable pathology involved primarily the macula, and both the full-field ERG and visual fields were normal. Acute CNV occurred during the third through fifth decades, with second eyes typically also affected during the subsequent year. Three affected members complained of nyctalopia prior to developing CNV. A Ser-181-Cys mutation in the TIMP-3 gene cosegregated with CNV in 10 affected subjects but was absent in 3 relatives at risk and sufficiently old to trust the clinical designation of normalcy. Nine eyes of 6 family members were treated by laser photocoagulation by 5 different ophthalmologists for foveal and juxtafoveal CNV. All eyes had recurrent CNV and lost acuity to 20/300 or less within several months. CONCLUSIONS: Laser photocoagulation of CNV did not stem vision loss in this SFD family. Although possible benefits of laser treatment were not put to formal clinical trial owing to the limited number of Sorsby's cases, it appears that photocoagulation is not of long-term benefit for preserving vision loss from the TIMP-3 Ser-181-Cys mutation. Several younger family members with the mutation are thus far not clinically affected and are being followed up.

Adult↗

Phylogenetic relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA, 28S rDNA D2, 18S rDNA gene sequences and morphological characters.

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA, 28S rDNA D2 region, and 18S rDNA gene sequences and morphological data using both PAUP* 4.0 and MRBAYES 3.0B4 from 88 in-group taxa representing 35 subfamilies. The monophyletic nature of almost all subfamilies, of which multiple representatives are present in this study, is well-supported except for two subfamilies, Cenocoelinae and Neoneurinae that should probably be treated as tribal rank taxa in the subfamily Euphorinae. The topology of the trees generated in the present study supported the existence of three large generally accepted lineage or groupings of subfamilies: two main entirely endoparasitic lineages of this family, referred to as the "helconoid complex" and the "microgastroid complex," and the third "the cyclostome." The Aphidiinae was recovered as a member of the non-cyclostomes, probably a sister group of Euphorinae or Euphorinae-complex. The basal position of the microgastroid complex among the non-cyclostomes has been found in all our analyses. The cyclostomes were resolved as a monophyletic group in all analyses if two putatively misplaced groups (Mesostoa and Aspilodemon) were excluded from them. Certain well-supported relationships evident in this family from the previous analyses were recovered, such as a sister-group relationships of Alysiinae+Opiinae, of Braconinae+Doryctinae, and a close relationship between Macrocentrinae, Xiphozelinae, Homolobinae, and Charmontinae. The relationships of "Ichneutinae + ((Adeliinae + Cheloninae) + (Miracinae + (Cardiochilinae + Microgastrinae)))" was confirmed within the microgastroid complex. The position of Acampsohelconinae, Blacinae, and Trachypetinae is problematic.

Animals↗