Evidence for gene flow differs from observed dispersal patterns in the Humboldt penguin, Spheniscus humboldti

The Humboldt penguin, once common throughout its range, is today listed as Vulnerable by the IUCN. Mark-recapture and telemetry studies indicate that adult Humboldt penguins are sedentary, suggesting strong genetic differentiation between colonies. We developed genotypes for 336 individuals at 12 microsatellite loci sampled at four different localities spanning the entire range of this species. Results show that long-term gene flow has occurred but appears to be affected by geographic distance as pairwise F _ST comparisons involving the colony at Punta San Juan (Peru) and the two colonies at Algarrobo (central Chile) and Puñihuil (southern Chile) are significant. Bayesian estimates of recent migration rates indicate substantial dispersal among all colonies. Despite the dramatic decline in numbers, we did not observe a bottleneck in any population. Furthermore, we did not detect a founder effect in the recently discovered colony at Puñihuil. As our indirect estimates signal strong gene flow between populations, we suggest that Humboldt penguin colonies need to be managed as a metapopulation rather than as discrete management units.     

The Mhc class II of the Black grouse (Tetrao tetrix) consists of low numbers of B and Y genes with variable diversity and expression

We found that the Black grouse (Tetrao tetrix) possess low numbers of Mhc class II B (BLB) and Y (YLB) genes with variable diversity and expression. We have therefore shown, for the first time, that another bird species (in this case, a wild lek-breeding galliform) shares several features of the simple Mhc of the domestic chicken (Gallus gallus). The Black grouse BLB genes showed the same level of polymorphism that has been reported in chicken, and we also found indications of balancing selection in the peptide-binding regions. The YLB genes were less variable than the BLB genes, also in accordance with earlier studies in chicken, although their functional significance still remains obscure. We hypothesize that the YLB genes could have been under purifying selection, just as the mammal Mhc-E gene cluster.     

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus)

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species. The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB301478, AB301944–AB301950, AB302087–AB302090, AB302190–AB302192, AB302843, AB302844, and AB303942–AB303945.     

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

Antigen-specific CD4⁺ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4⁺ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4⁺ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4⁺ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4⁺ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4⁺ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4⁺ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.     

Influence of dietary vitamins A and E on serum alpha- and gamma-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti)

Serum retinol, retinyl palmitate, beta-carotene, cryptoxanthin, lutein, alpha-tocopherol and gamma-tocopherol were measured in 18 captive Humboldt penguins (Spheniscus humboldti) prior to and following the removal of Columbia River (CR) smelt (Thaleichthys pacificus) from the diet. Dietary vitamin A was reduced from 59.8 to 13.5 IU g-1 (dry matter basis) when CR smelt was removed from the diet. Minimal changes were noted in dietary vitamin E. Serum samples Without-CR smelt had significantly lower circulating retinol (1.19 +/- 0.09 vs. 1.94 +/- 0.08 micrograms ml-1) and retinyl palmitate (0.033 +/- 0.012 vs. 0.105 +/- 0.004 microgram ml-1) compared to samples With-CR. The Without-CR smelt diet resulted in increased serum alpha-tocopherol from 26.4 +/- 0.94 to 39.1 +/- 3.72 micrograms ml-1. More serum samples taken Without-CR smelt had detectable levels of gamma-tocopherol than those With-CR smelt. Serum lutein was higher for the samples taken Without versus With-CR smelt. Serum cryptoxanthin did not differ. beta-Carotene was not detected. Data indicate that high levels of dietary vitamin A can affect circulating levels of retinol, retinyl palmitate and vitamin E. Thus, dietary vitamin A and the interrelationship between vitamins A and E should be considered when assessing captive penguins.     

Seasonal variations in the behavioural thermoregulation of roosting Humboldt penguins ( Spheniscus humboldti) in north-central Chile

We examined the thermoregulatory behaviour (TRB) of roosting Humboldt penguins (Spheniscus humboldti) in north central Chile during summer and winter, when ambient temperatures (T_a) are most extreme. Each body posture was considered to represent a particular TRB, which was ranked in a sequence that reflected different degrees of thermal load and was assigned an arbitrary thermoregulatory score. During summer, birds exhibited eight different TRBs, mainly oriented to heat dissipation, and experienced a wide range of T_a (from 14 to 31°C), occasionally above their thermoneutral zone (TNZ, from 2 to 30°C), this being evident by observations of extreme thermoregulatory responses such as panting. In winter, birds exhibited only three TRBs, mainly oriented to heat retention, and experienced a smaller range of T_a (from 11 to 18°C), always within the TNZ, even at night. The components of behavioural responses increased directly with the heat load which explains the broader behavioural repertoire observed in summer. Since penguins are primarily adapted in morphology and physiology to cope with low water temperatures, our results suggest that behavioural thermoregulation may be important in the maintenance of the thermal balance in Humboldt penguins while on land.     

Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n = 200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n = 195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n = 5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2–TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.     

PB1样铜绿假单胞菌噬菌体PHW2的生物学特性

【背景】铜绿假单胞菌(Pseudomonas aeruginosa)是一种引起医院感染、急性感染和慢性感染的常见条件致病菌。多重耐药铜绿假单胞菌仍然是引起严重医院感染的常见病菌,其临床治疗面临严峻挑战。噬菌体具有特异性杀菌的能力,在防治铜绿假单胞菌耐药菌方面具有应用前景。【目的】分离能裂解碳青霉烯类耐药的铜绿假单胞菌的噬菌体,分析其生物学特性和基因组特征,为噬菌体治疗储备资源。【方法】采集环境水样,用双层琼脂平板法分离噬菌体,对其形态、一步生长曲线、感染复数等生物学特性进行研究;使用IlluminaMiSeq平台测定噬菌体的全基因组序列,利用Newbler3.0、GeneMarkS、BLASTp、Mauve2.4.0等生物信息软件进行拼接、注释和比较基因组学分析。【结果】分离到一株噬菌体PHW2,该噬菌体属肌尾病毒科成员,可裂解7株碳青霉烯类耐药的铜绿假单胞菌;其最佳感染复数为0.1。一步生长曲线结果显示,其感染宿主菌PA001的潜伏期为100 min,裂解期为360 min,裂解量为25 PFU/cell;噬菌体PHW2在温度25-50℃和pH 6.0-8.0范围内稳定;紫外照射7 ...     

Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage

The knowledge of molecular alterations in osteoarthritic cartilage is important to identify novel therapeutic targets or to develop new diagnostic tools. We aimed to characterize the molecular response to cartilage degeneration by identification of differentially expressed genes in human osteoarthritic versus normal cartilage. Gene fragments selectively amplified in osteoarthritic cartilage by cDNA representational difference analysis included YKL-39 and the oesophageal-cancer-related-gene-4 (ECRG4). YKL-39 expression was significantly upregulated in cartilage from patients with osteoarthritis (n=14) versus normal subjects (n=8) according to real-time PCR (19-fold, p=0.009) and cDNA array analysis (mean 15-fold, p<0.001) and correlated with collagen 2 up-regulation. In contrast, the homologous cousin molecule YKL-40 (chitinase 3-like 1), which is elevated in serum and synovial fluid of patients with arthritis, showed no significant regulation in OA cartilage. Enhanced levels of YKL-40 may, therefore, be derived from synovial cells while modulation of YKL-39 and collagen 2 expression reflected the cartilage metabolism in response to degradation.     

Expression of ASK1-like genes in arrested stamens of female Silene latifolia plants

Silene latifolia is a dioecious plant in which sex is determined by heteromorphic sex chromosomes. In female plants, stamen development is arrested before microspore mother cells are formed. In this study, we isolated four cDNAs (SlSKP1-1 to 4) encoding ASK1-like protein as expression markers to reveal when expression levels are reduced in arrested stamens of female flowers. Expression patterns of the SlSKP1 genes were analyzed by in-situ hybridization. We use the flower development classification of Grant et al. (in Plant J 6:471–480, 1994). SlSKP1 genes were highly expressed in primary parietal cells and primary sporogenous cells that develop into microspore mother cells in male flowers. Expression levels started to be reduced in the external stamens of the female flowers when stamen development was arrested at stage 7. Although microspore mother cells could not be developed in female flowers and SlSKP1 expression may be unnecessary in arrested stamens, SlSKP1 genes were still expressed in sporogenous cells of degenerated stamens at stage 8. Parietal cells stopped differentiating earlier than sporogenous cells in arrested stamens. These results suggest that not all types of cell are arrested simultaneously at a particular stage of stamen development during stamen suppression of S. latifolia.     

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae)

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.     

Recent duplication and inter-locus gene conversion in major histocompatibility class II genes in a teleost,the three-spined stickleback

Using a bacterial artificial chromosome (BAC) library, we analysed a 99.5 kb genomic segment containing the major histocompatibility class II genes of a teleost, the three-spined stickleback Gasterosteus aculeatus. Experiments with G. aculeatus have provided direct evidence for balancing selection by pathogens and mate choice driving MH class II beta polymorphism. Two sets of paralogous class II alpha genes and beta genes in a tandem arrangement were identified, designated Gaac-DAA/DAB and Gaac-DBA/DBB. Expression analysis of the beta genes using single-strand conformation polymorphism revealed that both gene copies are expressed. Based on an analysis of pairwise nucleotide polymorphisms, we estimate that the duplication into two paralogous class II loci occurred only 1.2–2.4 million years ago, 1–2 orders of magnitude more recently than in other fish, bird or mammalian species. At the 3-direction of the classical MH loci, we identified another seven genes or gene fragments, two of which (small inducible cytokine, complement regulatory factor) are related to immune function in other vertebrates. None of these genes were associated with MH class II genes in zebrafish, suggesting a markedly different organisation of the MH class II region in sticklebacks, and presumably, across bony fishes. When the nucleotide substitution pattern of the novel class II beta genes was analysed together with a representative sequence sample isolated from fish in northern Germany (n=27), we found that the peptide binding region of the Gaac-DAB and Gaac-DBB loci had undergone an inter-locus gene conversion (P=0.007). In accordance, we found a 10- to 20-fold higher frequency of CpG-islands on the MH class II segment compared to other species, a feature that may be conducive for inter-locus recombination.     

Duplication polymorphism at MHC class II DRB1 locus in the wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>)

The origin of allelic polymorphism in genes of the major histocompatibility complex represents a central topic in evolutionary genetics as it is probably the most polymorphic region in the nuclear genome of vertebrates. Accordingly, the analyses of genetic variability at these loci provide evidence complementary to the population genetics studies based on neutral loci. In this study, four wild boar populations, two from Italy (Florence region and Castelporziano Presidential Reserve, outside Rome) and one each from Hungary and Poland, were characterized at a highly polymorphic fragment including part of intron 1 and exon 2 of swine leukocyte antigen (SLA) class II DRB1 gene by direct sequencing and by cloning. Excluding the false alleles, a total of 18 different sequences were observed in 57 individuals. The high ratio of nonsynonymous (dN) vs synonymous (dS) substitution rates in the peptide-binding region supports the hypothesis that balancing selection is operating at this locus. A duplication event at the DRB1 gene was documented only in one Italian population with both copies being putatively active. This is the first evidence of a polymorphism for the number of copies of an SLA gene.     

Potential problems in estimating the male-to-female mutation rate ratio from DNA sequence data

It is commonly believed that the rate of mutation is much higher in males than in females because the number of germ-cell divisions per generation is much larger in males than in females. However, the precise magnitude of the male-to-female mutation rate ratio (α _m ) remains unknown. Recently there have been efforts to estimate α _m by using DNA sequence data from different species. We have studied the potential problems in such an approach. We found that the rate of synonymous substitution varies about fivefold among X-linked genes, as large as the variation among autosomal genes. This large variation makes the assumption of selective neutrality of synonymous changes dubious, so one should be cautious in using the synonymous rates in X-linked and autosomal genes to estimate α _m . A similar difficulty was also observed in using nonhomologous intron sequences to estimate α _m . Contrary to the expectation that X-linked sequences should evolve more slowly than autosomal sequences, theAlu repeat in the last intron of the X-linked zinc finger gene has evolved faster than the four autosomalAlu repeats used in this study. It appears that the best way to estimate α _m is to use homologous sequences. However, such sequences may be involved in gene conversion events. In fact, we found evidence that the Y-linked and X-linked zinc finger genes have been involved in multiple conversion events during primate evolution. Thus, the possibility of gene conversion should be considered when using homologous sequences to estimate α _m . Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992