The natural autoantibody repertoire of nonobese diabetic mice is highly active

Analysis of spontaneous hybridomas generated from nonobese diabetic (NOD) mice indicates that the natural autoantibody repertoire of NOD mice is highly active compared with C57BL/6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. Even when selected for binding to a prototypic beta cell Ag, such as insulin, NOD mAb have characteristics of natural autoantibodies that include low avidity and broad specificity for multiple Ags. Analyses of the variable region of Ig H chain (V(H)) and variable region kappa L chain genes expressed by six insulin binding mAb show that V gene segments are often germline encoded and are identical with those used by autoantibodies, especially anti-dsDNA, from systemic autoimmune disease in MRL, NZB/W, and motheaten mice. V(H) genes used by four mAb are derived from the large J558 family and two mAb use V(H)7183 and V(H)Q52 genes. The third complementarity-determining region of Ig H chain of these mAb have limited N segment diversity, and some mAb contain DNA segments indicative of gene replacement. Genetic abnormalities in the regulation of self-reactive B cells may be a feature that is shared between NOD and conventional systemic autoimmune disorders. In NOD, the large pool of self-reactive B cells may fuel autoimmune beta cell destruction by facilitating T-B cell interactions, as evidenced by the identification of one mAb that has undergone Ag-driven somatic hypermutation.     

Immunoglobulin genomics in the guinea pig (Cavia porcellus)

In science, the guinea pig is known as one of the gold standards for modeling human disease. It is especially important as a molecular and cellular biology model for studying the human immune system, as its immunological genes are more similar to human genes than are those of mice. The utility of the guinea pig as a model organism can be further enhanced by further characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the guinea pig immunoglobulin (Ig) heavy and light chain genes. The guinea pig IgH locus is located in genomic scaffolds 54 and 75, and spans approximately 6,480 kb. 507 V(H) segments (94 potentially functional genes and 413 pseudogenes), 41 D(H) segments, six J(H) segments, four constant region genes (μ, γ, ε, and α), and one reverse δ remnant fragment were identified within the two scaffolds. Many V(H) pseudogenes were found within the guinea pig, and likely constituted a potential donor pool for gene conversion during evolution. The Igκ locus mapped to a 4,029 kb region of scaffold 37 and 24 is composed of 349 V(κ) (111 potentially functional genes and 238 pseudogenes), three J(κ) and one C(κ) genes. The Igλ locus spans 1,642 kb in scaffold 4 and consists of 142 V(λ) (58 potentially functional genes and 84 pseudogenes) and 11 J(λ) -C(λ) clusters. Phylogenetic analysis suggested the guinea pig's large germline V(H) gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves.     

Similarities and differences between the light and heavy chain Ig variable region gene repertoires in chronic lymphocytic leukemia

Analyses of Ig V(H)DJ(H) rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V(L) and J(L) gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V(L) genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V(L) and J(L) segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V(H) repertoire, V(L) gene use was not significantly different than that of normal B-lymphocytes. In addition, Vkappa genes that lie more upstream on the germline locus were less frequently mutated than those at the 3' end of the locus; this was not the case for Vlambda genes and is not for V(H) genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.     

Marsupial light chains: IGK with four V families in the opossum Monodelphis domestica

 A full-length and several partial cDNAs encoding IGK light chains from the marsupial South American opossum, Monodelphis domestica, were isolated and characterized. Using these clones as a starting point, the expressed IGKV repertoire was sampled by anchored polymerase chain reaction using an IGKC-specific primer. Based on nucleotide sequences of twenty unique, expressed IGKV-J combinations, there are at least four IGKV families and two J segments. Southern blot analysis revealed each IGK-V family contains multiple gene segments totaling at least thirty-five IGKV in the opossum genome. No evidence for particular, recurrent IGKV-J combinations in the opossum IGK repertoire was seen, rather the V-J combinations appeared random and diverse. Each of the four IGKV families appear more closely related to V segments from placental mammals than to each other, suggesting the duplication of the IGKV families prior to the separation of marsupials and placental mammals more than one-hundred-million years ago. Overall, the complexity of opossum light chain V segments appears greater than that found in the heavy chain, and light chains are likely to contribute significantly to Ig diversity in this species.With this report, the homologues encoding all three classes of eutherian Ig chains, IGH, IGL, and IGK, have been described in a non-placental mammal. Received: 5 April 1999 / Revised: 3 June 1999     

Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire

The antigen-binding site of the camel heavy-chain antibodies devoid of light chain consists of a single variable domain (V(H)H) that obviously lacks the V(H)-V(L) combinatorial diversity. To evaluate the extent of the V(H)H antigen-binding repertoire, a germline database was constructed from PCR-amplified V(H)H/V(H) segments of a single specimen of Camelus dromedarius. A total of 33 V(H)H and 39 V()H unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the V(H)Hs evolved within the V(H) subgroup III. Nevertheless, the V(H)H germline segments are highly diverse, leading to a broad structural repertoire of the antigen-binding loops. Seven V(H)H subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged V(H)Hs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the V(H)H germline genes at the regions affecting the structure of the antigen-binding loops.     

Constraints on the morphological evolution of marsupial shoulder girdles

Throughout their evolutionary histories, marsupial mammals have been taxonomically and morphologically less diverse than their sister taxa the placentals. Because of this, it has been proposed that the evolution of marsupials has been constrained by the functional requirements of their mode of reproduction. Marsupials give birth after short gestation times to immature neonates that immediately crawl, under the power of their precociously developed shoulder girdles, to the teat where they attach and complete their early development. Using a novel approach incorporating adult and embryological morphological data, this study is the first to both: (1) statistically support adult patterns of morphological divergence consistent with the constraint hypothesis, and (2) identify ontogenetic patterns of morphological change that demonstrate that the constraint was responsible, at least in part, for their formation. As predicted by the marsupial constraint, the shoulder girdles of adult marsupials are less diverse than those of adult placentals, and adult marsupial scapulae are less morphologically diverse than adult marsupial pelves. Furthermore, marsupials that complete an extensive crawl to the teat are restricted to a common pattern of ontogenetic scapular shape change, strongly supporting the hypothesis that the morphological development of the marsupial scapula has been limited evolutionarily by its obligate role in the crawl to the teat. Because this study establishes that ontogenetic and evolutionary morphological change is correlated within mammalian scapulae, it is probable that the marsupial constraint also restricted the morphological divergence of the scapula over evolutionary time by limiting ontogenetic change in the scapula. These findings, coupled with the importance of the shoulder girdle in mammalian locomotor specialization, support the conclusion that the low morphological diversity of marsupial forms over evolutionary time could be directly due to the constraint on marsupial morphological evolution caused by the functional requirements of the crawl to the teat.     

Somatic hypermutation and junctional diversification at Ig heavy chain loci in the nurse shark

We estimate there are approximately 15 IgM H chain loci in the nurse shark genome and have characterized one locus. It consists of one V, two D, and one J germline gene segments, and the constant (C) region can be distinguished from all of the others by a unique combination of restriction endonuclease sites in Cmu2. On the basis of these Cmu2 markers, 22 cDNA clones were selected from an epigonal organ cDNA library from the same individual; their C region sequences proved to be the same up to the polyadenylation site. With the identification of the corresponding germline gene segments, CDR3 from shark H chain rearrangements could be analyzed precisely, for the first time. Considerable diversity was generated by trimming and N addition at the three junctions and by varied recombination patterns of the two D gene segments. The cDNA sequences originated from independent rearrangements events, and most carried both single and contiguous substitutions. The 53 point mutations occurred with a bias for transition changes (53%), whereas the 78 tandem substitutions, mostly 2-4 bp long, do not (36%). The nature of the substitution patterns is the same as for mutants from six loci of two nurse shark L chain isotypes, showing that somatic hypermutation events are very similar at both H and L chain genes in this early vertebrate. The cis-regulatory elements targeting somatic hypermutation must have already existed in the ancestral Ig gene, before H and L chain divergence.     

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

Why are There Fewer Marsupials than Placentals? On the Relevance of Geography and Physiology to Evolutionary Patterns of Mammalian Diversity and Disparity

Placental mammals occupy a larger morphospace and are taxonomically more diverse than marsupials by an order of magnitude, as shown by quantitative and phylogenetic studies of several character complexes and clades. Many have suggested that life history acts as a constraint on the evolution of marsupial morphology. However, the frequent circumvention of constraints suggests that the pattern of morphospace occupation in marsupials is more a reflection of lack of ecological opportunity than one of biases in the production of variants during development. Features of marsupial physiology are a potential source of biases in the evolution of the group; these could be coupled with past macroevolutionary patterns that followed conditions imposed by global temperature changes. This is evident at the K/Pg boundary and at the Eocene/Oligocene boundary. The geographic pattern of taxonomic and morphological diversity in placental clades mirrors that of extant placentals as a whole versus marsupials: placentals of northern origin are more diverse those of southern one and include the clades that are outliers in taxonomic (rodents and bats) and ecomorphological (whales and bats) richness.     

Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.     

Thermogenic capabilities of the opossum Monodelphis domestica when warm and cold acclimated: similarities between American and Australian marsupials

1. Monodelphis domestica is a small marsupial mammal from South America. Its thermogenic abilities in the cold were determined when the opossums were both warm (WA) and cold (CA) acclimated. Maximum heat production of M. domestica was obtained at low temperatures in helium-oxygen. 2. Basal metabolic rate (BMR) in the WA animals was 3.2 W/kg and mean body temperature was 32.6 degrees C at 30 degrees C. These values were lower than those generally reported for marsupials. Nevertheless, these M. domestica showed considerable metabolic expansibility in response to cold. Sustained (summit) metabolism was 8-9 times BMR, while peak metabolism was 11-13 times BMR. These maximum values were equal to, or above, those expected in small placentals. 3. Cold acclimation altered the thermal responses of M. domestica, particularly in warm TaS. However, summit metabolism was not significantly increased; nor did M. domestica show a significant thermogenic response to noradrenaline, which in many small placentals elicits non-shivering thermogenesis. The thermoregulatory responses of this American marsupial were, in most aspects, similar to those of Australian marsupials. This suggests that the considerable thermoregulatory abilities of marsupials are of some antiquity.     

Mapping human X-linked genes in the phalangerid marsupial Trichosurus vulpecula.

We mapped 15 human X-chromosome markers in the common brush-tailed possum, Trichosurus vulpecula (Kerr), which represents the Australian marsupial family Phalangeridae. In situ hybridization was used to localize highly conserved human X-linked genes to chromosomes of T. vulpecula diploid lines. Ten genes located on the long arm of the human X (human Xq genes) all mapped to the possum X chromosome. However, all five genes located on the short arm of the human X (human Xp genes) mapped to autosomes. These findings confirm our previous work, which showed that the X chromosome in macropodid and dasyurid marsupials bears all the human Xq genes but none of the human Xp genes studied. This suggests that the marsupial X is highly conserved, but its gene content reflects that of only part of the eutherian X, a result consistent with our hypothesis that an autosomal region was added to the X early in eutherian divergence.     

Partitioning of rearranged Ig genes by mutation analysis demonstrates D-D fusion and V gene replacement in the expressed human repertoire

The accurate partitioning of Ig H chain V(H)DJ(H) junctions and L chain V(L)J(L) junctions is problematic. We have developed a statistical approach for the partitioning of such sequences, by analyzing the distribution of point mutations between a determined V gene segment and putative Ig regions. The establishment of objective criteria for the partitioning of sequences between V(H), D, and J(H) gene segments has allowed us to more carefully analyze intervening putative nontemplated (N) nucleotides. An analysis of 225 IgM H chain sequences, with five or fewer V mutations, led to the alignment of 199 sequences. Only 5.0% of sequences lacked N nucleotides at the V(H)D junction (N1), and 10.6% at the DJ(H) junction (N2). Long N regions (>9 nt) were seen in 20.6% of N1 regions and 17.1% of N2 regions. Using a statistical analysis based upon known features of N addition, and mutation analysis, two of these N regions aligned with D gene segments, and a third aligned with an inverted D gene segment. Nine additional sequences included possible alignments with a second D segment. Four of the remaining 40 long N1 regions included 5' sequences having six or more matches to V gene end motifs, which may be the result of V gene replacement. Such sequences were not seen in long N2 regions. The long N regions frequently seen in the expressed repertoire of human Ig gene rearrangements can therefore only partly be explained by V gene replacement and D-D fusion.     

Unexpected oocyte growth after follicular antrum formation in four marsupial species.

During examination of maturing preovulatory marsupial oocytes we noted that oocyte diameters were invariably about 50% greater than the figures reported in earlier histological studies. As all previous investigations were limited to small follicles (at most 25% the size of the ovulating follicle), the present study was initiated to examine oocyte growth during the whole period of follicular development. Oocyte and follicle diameters were measured for three Australian (Trichosurus vulpecula, Macropus eugenii and Bettongia penicillata--fresh nonfixed material) and one American marsupial species (Monodelphis domestica--histological sections) in which multiple follicle development had been induced by exogenous gonadotrophin treatment. In all species oocytes were obtained from follicles ranging from pre-antral to immediately pre-ovulatory (maximum follicle sizes obtained were: T. vulpecula, 4.5 mm; M. eugenii, 4.3 mm; B. penicillata, 2.5 mm; M. domestica, 0.7 mm). In two of the species (T. vulpecula and B. penicillata) ovulated oocytes were also examined. In T. vulpecula and M. eugenii oocytes were found to achieve much greater diameters than previously reported from histological studies of small follicles (< 0.8 mm) and similar patterns of growth were found in the other two species. In the four species oocytes reached diameters about two to three times that found for eutherian mammals. It was concluded that the marsupial oocyte continued to grow after formation of the follicular antrum and that, although the rate of oocyte growth slowed in larger follicles, it continued into the period immediately before ovulation. In B. penicillata the largest oocytes were obtained after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carpal ontogeny in Monodelphis domestica and Caluromys philander (Marsupialia)

Carpal bones have experienced numerous changes during marsupial evolution, even though their diversity and development remain poorly studied. The aim of this work was to document adult form and the pattern of mesenchymal tissue condensation and formation of chondrification and ossification centers in the hand of two marsupials. Two fundamental questions were asked: whether the loss of embryonic precursors was associated with the loss of adult elements, or whether there were developmental signs of ancestral mammalian elements that have been fused or lost in marsupial taxa. We were also interested to find out whether there is sexual dimorphismus in the carpals, as has been reported for some didelphids. Histological sections, cleared and stained specimens and macerated skeletons representing an ontogenetic series of Monodelphis domestica were used to document carpal development. Comparisons were made with perinatal stages of Caluromys philander and with adult specimens of other marsupials. A prenatal M. domestica in the 13th day after conception has a cell condensation that because of its position is homologized with a centrale, which is at birth already lost or fused. Neonatal M. domestica and C. philander have the number and arrangement of their adult carpal anatomy. Trapezium and trapezoid start ossification later than most other carpals, while pisiform and prepollex are the last to do so. Adult males of M. domestica have relatively larger and more robust pisiforms, compared to other carpals, than females. This sexual dimorphism develops relatively late as it was not recorded in male specimens around 160 days old. An extra sesamoid bone located just distal to the radius and proximo-palmar to the scaphoid was recorded in specimens of C. philander, C. derbianus and Didelphis virginiana.