The complete mitochondrial genome of the hagfish Myxine glutinosa: unique features of the control region.

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism.

The complete nucleotide sequence of the mitochondrial DNA of the agnathan Lampetra fluviatilis: bearings on the phylogeny of cyclostomes.

There are two competing theories about the interrelationships of craniates: the cyclostome theory assumes that lampreys and hagfishes are a clade, the cyclostomes, whose sister group is the jawed vertebrates (gnathostomes); the vertebrate theory assumes that lampreys and gnathostomes are a clade, the vertebrates, whose sister group is hagfishes. The vertebrate theory is best supported by a number of unique anatomical and physiological characters. Molecular sequence data from 18S and 28S rRNA genes rather support the cyclostome theory, but mtDNA sequence of Myxine glutinosa rather supports the vertebrate theory. Additional molecular data are thus needed to elucidate this three-taxon problem. We determined the complete nucleotide sequence of the mtDNA of the lamprey Lampetra fluviatilis. The mtDNA of L. fluviatilis possesses the same genomic organization as Petromyzon marinus, which validates this gene order as a synapomorphy of lampreys. The mtDNA sequence of L. fluviatilis was used in combination with relevant mtDNA sequences for an approach to the hagfish/lamprey relationships using the maximum-parsimony, neighbor-joining, and maximum-likelihood methods. Although trees compatible with our present knowledge of the phylogeny of craniates can be reconstructed by using the three methods, the data collected do not support the vertebrate or the cyclostome hypothesis. The present data set does not allow the resolution of this three-taxon problem, and new kinds of data, such as nuclear DNA sequences, need to be collected.

Immune-mediated destruction of melanocytes in halo nevi is associated with the local expansion of a limited number of T cell clones.

The beta-chain repertoire of the T cells that infiltrate spontaneously regressing nevi (the halo nevus phenomenon) was studied. In addition to the infiltration of the halo nevi by cutaneous lymphocyte-associated Ag-positive lymphocytes, oligoclonal expansion of T cells was observed in all halo nevi of all patients. T cells using the same TCR beta-chain were observed in distinct halo nevi of the same patient but not in his peripheral blood, demonstrating a local expansion of common clones that are most likely activated by the Ag(s) shared by independent halo nevi of the same patient.

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative.

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

The complete nucleotide sequence of the mitochondrial DNA of the dogfish, Scyliorhinus canicula.

We have determined the complete nucleotide sequence of the mitochondrial DNA (mtDNA) of the dogfish, Scyliorhinus canicula. The 16,697-bp-long mtDNA possesses a gene organization identical to that of the Osteichthyes, but different from that of the sea lamprey Petromyzon marinus. The main features of the mtDNA of osteichthyans were thus established in the common ancestor to chondrichthyans and osteichthyans. The phylogenetic analysis confirms that the Chondrichthyes are the sister group of the Osteichthyes.

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates.

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.

The main features of the craniate mitochondrial DNA between the ND1 and the COI genes were established in the common ancestor with the lancelet.

We have cloned the mitochondrial DNA fragment extending from tRNA-Leu to the cytochrome oxidase subunit 1 (COI) genes of Branchiostoma lanceolatum, Myxine glutinosa, Lampetra fluviatilis, and Scyliorhinus caniculus and have determined their respective gene sequences and organization. In all four species, this region contains the ND1 and ND2 genes and the genes coding eight tRNAs, namely, tRNA-Ile, -Gln, -Met, -Trp, -Ala, -Asn, -Cys, and -Tyr. The gene order is the same in the hagfish, lamprey and dogfish. In the lancelet, the location of the tRNA genes is slightly different. The mitochondrial code of Myxine, Lampetra, and Scyliorhinus is identical to that of vertebrates. The code used by the lancelet is the same with the exception of AGA (a stop codon in vertebrates), which codes for glycine in the lancelet. From the comparison of the four maps with already published ones for other species, we propose that the main features of the craniate mtDNA between the ND1 and COI genes were established in the common ancestor to cephalochordates and vertebrates more than 400 MYA. The origin of replication of the light-strand (Ori-L), usually located between the tRNA-Asn and tRNA-Cys genes in vertebrates, was not found in the lancelet, hagfish, or lamprey (Lampetra). In contrast, it was found in the dogfish. Thus the position of Ori-L was established for the first time in the common ancestor to the Chondrichthyes and Osteichthyes and remained present in all later-emerging vertebrates.

A unique cDNA coding for subunits 8 and 6 of mitochondrial adenosine triphosphatase of the lancelet Branchiostoma lanceolatum, an ancestor of vertebrates.

A cDNA encoding subunits 8 and 6 of mitochondrial ATPase of the cephalochordate lancelet (Branchiostoma lanceolatum), a direct ancestor of vertebrates, has been cloned and sequenced. A unique transcript encodes the 8 and 6 subunits. In the course of isolation of the 5' end of the ATPase 8 subunit gene, we also determined the sequence of the 5' adjacent tRNA-Lys gene. The anticodon used by mitochondrial tRNA-Lys is TTT.

Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.

Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice.

We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.

Major histocompatibility complex class I molecules and resistance against intracellular pathogens.

The immune system employs a temporal hierarchy of effector mechanisms to combat infections by intracellular pathogens. The nonspecific response is independent of MHC and can be activated rapidly, while the specific response is slower, more specific, and requires major histocompatibility complex (MHC) molecules. MHC-dependent responses have been characterized extensively in vitro for antigens presented by polymorphic MHC class Ia and class II proteins and recognized by T lymphocytes carrying alpha/beta T-cell receptors (TcR). Growing indirect evidence has implicated monomorphic MHC class Ib proteins and gamma/delta T lymphocytes in defense against bacterial infections, but the biochemical and immunological behavior of class Ib proteins and gamma/delta TcR has not been well characterized, and most hypotheses involving these proteins have relied on data obtained with polymorphic MHC proteins and alpha/beta TcR. An overview of studies describing bacterial infections in vivo suggests that, in many cases, MHC class I-dependent effector cells may not be indispensable for effective immune responses, exerting instead a modulatory effect during the course of infection. Furthermore, many class Ib proteins have probably specialized to present stress antigens and conserved microbial antigens, which may be recognized by gamma/delta T cells through an interaction that is qualitatively very different from alpha/beta TcR binding to class I and class II proteins.

Duplication of the CD8 beta-chain gene as a marker of the man-gorilla-chimpanzee clade.

In earlier studies we have found that the gene encoding the CD8 beta chain is duplicated in man. We demonstrate here that the duplicated genes are both located on chromosome 2. We have also studied the moment of the duplication event relative to the evolution of higher primates by using genomic DNA of a panel of primates. Our data strongly suggest that duplication occurred after the orangutan lineage had split and before the chimpanzee, gorilla, and man clade diverged, some 8-9.5 million years ago. This result makes the CD8 beta-chain gene duplication a convenient marker for the study of the evolution of higher primates.

Evolution of the mouse t haplotype: recent and worldwide introgression to Mus musculus.

Mouse t haplotypes are variants of chromosome 17, consisting of four inversions. Despite the homozygous lethality and pleiotropic effect on embryonic development, sperm production, and recombination, they have widely spread in natural populations of the house mouse (10-40% in frequency) because of the meiotic drive advantage. We sequenced 14 Tcp-1 (t-complex polypeptide 1) genes from four t haplotypes, nine wild mice, and a rat as a reference. From a comparison of intron sequences of 610 base pairs, we dated the origin of t haplotypes to 2.9 +/- 0.7 million years ago, which predates the splitting of Mus musculus subspecies (approximately 1 million years ago). However, the Tcp-1 intron sequences of t haplotypes from different M. musculus subspecies from various parts of the world show no divergence, indicating the recent introgression (no earlier than 0.8 million years ago) of a single ancestral type. Nucleotide changes in coding regions are also consistent with this conclusion. Hence, polymorphisms among t haplotypes including lethality factors have accumulated during this short time period independently in each M. musculus subspecies.

Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

Evolution of the major histocompatibility complex: a hundred-fold amplification of MHC class I genes in the African pigmy mouse Nannomys setulosus.

The genome of the African murine rodent Nannomys setulosus was found to harbor several thousand major histocompatibility complex (MHC) class I genes instead of the 30-40 genes found in conventional laboratory mice, which are mostly of Mus musculus domesticus origin. Other genes of N. setulosus, either functionally or physically linked to class I genes, are not amplified. Amplified genes derive from as few as three ancestors and amplification has likely occurred after the divergence of the two Nannomys species, N. setulosus and N. minutoides, which took place about three million years ago. Amplified genes are mostly pseudogenes. Statistical analysis of dinucleotide frequencies leads us to propose that inactivation of the genes has occurred through the repeat induced mutation process, a possible "newcomer" in the evolution of the MHC.

Suicide on the paternal and maternal sides of depressed patients with a lifetime history of attempted suicide.

The computational model of Slater, based on the analysis of ancestral secondary cases on the paternal and maternal sides of the subjects, was applied to depressed patients with a lifetime history of attempted suicide, either violent or nonviolent, in order to investigate possible modes of transmission of suicidal behavior. Among 549 patients, 15 had 2 or more ascendant first- and second-degree relatives who committed suicide. The results of the distribution of these cases were compatible with polygenic inheritance of suicidal behavior in depressed patients with a history of attempted suicide. In patients using violent methods, a significantly greater loading of ancestral secondary cases of suicide was observed on the maternal side and, in the nonviolent attempter group, on the paternal side.

Expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.

The murine beta 2-microglobulina cDNA was cloned into pAc373 and pVL941 transfer vectors and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. Both types of recombinant baculoviruses were isolated and used to infect Spodoptera frugiperda (Sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pVL941-derived virus than when the pAc373-based virus was used. beta 2m was secreted into the culture medium where it accumulated and, under the best conditions, reached an approximate level of 10 micrograms/10(6) cells. Pulse-chase experiments after metabolic labelling with 35S-methionine followed by immunoprecipitation showed that beta 2m was stable, but that the secretion process in infected cells was relatively slow. Recombinant beta 2m was endowed with biological activity and was indistinguishable from that produced by mouse cells in 2D gel analysis. beta 2m was purified to near homogeneity from serum-free culture medium conditioned by recombinant baculovirus-infected cells by using an immunoaffinity column. The use of the insect cell/baculovirus expression system should constitute a suitable source of mouse beta 2m and should aid experiments aimed at unraveling its interactions with mouse class I histocompatibility molecules.

Comparative structure of two duplicated T1a class I genes (T10c and 37) of the murine H-2d MHC. Implications on the evolution of the T1a region.

The class I Ag encoded in the Qa/T1a regions of the murine MHC are much less polymorphic, and usually have a more restricted tissue distribution than the classical histocompatibility class I Ag, encoded by genes located in the H-2K, D, and L loci. The isolation of a quasi-ubiquitously expressed, poorly polymorphic class I gene of the T1a region of the H-2d mouse MHC, namely gene 37 (or T18d), has been recently reported. We describe the nucleotide sequence of a closely related gene, T10c gene, the counterpart of the gene 37 in the large duplicated parts of T1a region of the BALB/c (H-2d) MHC. The T10c gene structure and sequence are very similar to those of gene 37, but T10c gene is most likely a pseudogene. In A/J mouse strain, there appears to be a single gene related to 37, which is also found expressed in a variety of tissues. We show that this gene is likely to be a chimeric one derived from T10c for its 3' part, and from a gene closely related to gene 37 for its 5' part, which potentially encodes for an unusual class I molecule composed of the first two domains. Finally, Southern blot analysis of a number of wild mice and related animals suggests that a gene closely related to the present T10c gene may be the ancestor of this subfamily of class I genes characterized by the presence of an unusual second domain.