Extreme Evolutionary Conservation of CaSox4 Gene* in Alligator sinensis*

The Sox genes form a gene family related by homology to the high-mobility-group (HMG) box region of the testis-determining gene, SRY.Using degenerate primer PCR, we have cloned and sequenced the HMG-box motif ofCaSox4 gene from genomic DNA of the Chinese alligator,Alligator sinensis, a species closely related to American alligator, Alligator mississippiensis. Sequence comparison showed that CaSox4 gene shared 97% homology at the nucleotide level and complete identity at the amino acid level, respectively, withAES4 gene of A. mississippiensis. It indicates that the Sox4gene has been extremely conserved during evolution. The result obtained in the present study strongly suggests that theSox4 gene did not diverge during the differentiation of two closely related alligators, which are thought to have originated from a common ancestor and have existed largely unchanged for 200–250 Myr.     

Molecular characterization and tissue expression profiles of insulin-like growth factor binding protein-1 (IGFBP-1) in Chinese alligator <Emphasis Type="Italic">Alligator sinensis</Emphasis> during the active and hibernating periods

The Chinese alligator Alligator sinensis (Fauvel, 1879) is an endangered species endemic to China. Up to this date, very little is known about the regulation of its growth and development. Insulin-like growth factor binding protein-1 (IGFBP-1) plays essential roles in regulating vertebrate growth and development by modulating IGFs action. In this study, we cloned the full-length cDNA of Chinese alligator IGFBP-1 (caIGFBP-1) for the first time, which has an open reading frame (ORF) of 885-bp nucleotides encoding a peptide of 294 amino acid residues. The caIGFBP-1 contains all features of IGFBP-1 peptide with the conserved cysteine-rich N- and C-terminal domains and variable central L-domain. Multiple alignment analysis showed that caIGFBP-1 shares high identity in amino acid sequence with the IGFBP-1 s of American alligator Alligator mississippiensis (Daudin, 1802) (99.32%) and birds (66.32–74.53%). Phylogenetic tree analysis of IGFBP-1 amino acid sequences indicated that alligators cluster into bird branch. The caIGFBP-1 is widely expressed in all the examined tissues, the expression level is highest in liver, higher in kidney, moderate in pancreas, stomach, small intestine, and lower in ovary, muscle, lung, heart. During the hibernating period, the expression level of caIGFBP-1 was significantly reduced in liver, kidney, stomach, pancreas and small intestine, while did not significantly change in heart, lung, ovary and muscle. The caIGFBP-1 mRNA expression changes during the active and hibernating periods, which coincides with that of Chinese alligator IGF, implicating that it might play important roles in the IGF-mediated growth and development of Chinese alligator.     

Gene structure and spatio-temporal expression of chicken LPIN2

LPIN2 is one of the members of the Lipin family, which acts as a phosphatidate phosphatase enzyme. In this study, we identified the cDNA sequence and exonic variants of chicken LPIN2, and evaluated its spatio-temporal expression patterns. It indicated that chicken LPIN2 cDNA contained a 2,664-bp open reading frame flanked by a 176-bp 5′ untranslated region and a 429-bp 3′ untranslated region, predicted encoding one protein of 886 amino acids. Fourteen variants (three missense mutations) were detected from the coding region of chicken LPIN2. W265L was predicted to affect the gene function (p < 0.01) and eight synonymous mutations were predicted to affect the binding sites of SR proteins, which suggested the important functions of these variants. Real-time quantitative PCR revealed that LPIN2 in two genotypic chickens (LD and HB chickens, with difference in growth rate) presented similar tissue expression patterns, which was liver and ovary enriched with low abundance in skeleton muscles. Chicken LPIN2 exhibited tissue-specific temporal-expression patterns during postnatal development (0–16 weeks). Chicken cutaneous LPIN2 was in steady-state mRNA levels during postnatal development; chicken LPIN2 mRNA in pectoralis major had a prominent level at 0 week-old, then dropped dramatically at 4 week-old and maintained a relatively low level through 4–16 weeks; while chicken hepatic LPIN2 had a relatively high expression at 0 week-old, with a relatively low level through 4–12 weeks and a slight increase at 16 week-old. The studies about the basic gene features of chicken LPIN2 would lay the foundation for further exploring its biological function.     

Extreme evolutionary conservation of CaSox4 gene in alligator Alligator sinensis

The SOX genes form a gene family related by homology to the high-mobility-group (HMG) box region of the testis-determining gene, SRY. Using degenerate primer PCR, we have cloned and sequenced the HMG-box motif of CaSox4 gene from genomic DNA of the Chinese alligator, Alligator sinensis, a species closely related to American alligator, Alligator mississippiensis. Sequence comparison showed that CaSox4 gene shared 97% homology at the nucleotide level and complete identity at the amino acid level, respectively, with AES4 gene of Alligator mississippiensis. It indicates that the Sox4 gene has been extremely conserved during evolution. The result obtained in the present study strongly suggests that the Sox4 gene did not diverge during the differentiation of two closely related alligators, which are thought to have originated from a common ancestor and have existed largely unchanged for 200-250 Myr.     

Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1–2 (n = 1) or exons 5–7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5′ and/or 3′ breakpoints located within Alu repeat elements. In addition, we identified the 5′ breakpoint of a previously reported deletion of exons 1–2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.     

扬子鳄的CaSox4基因的分子克隆和进化分析

The completely identical HMG-box motif of CaSox4 gene from both male and female genomic DNA of the Chinese alligator (alligator sinensis) was cloned and sequenced by degenerate primer PCR.Compared with the human and mouse SRY,CaSox4 revealed 51% and 57% nucleotide homology respectively and 49% and 55% amino acid identity respectively,CaSox4 belongs to subgroup C of the Sox gene family.The GC content is 86% in the HMG-box region of the CaSox4 gene,Blast analysis showed that the CaSox4 gene shares 100^ amino acid identity with human Sox4.bird SoxLF4,turtle Sra4 and lizard CvSox4 genes.Casox4 may be orthologous with the human SOx4 gene.This indicates that CaSox4 gene shows the remarkable evolutionary conservation during the evolution of Alligator Sinensis,The extensive sequence conservation of the Sox4 gene between reptiles,mammals and birds suggests major functional constraints[Acta Zoologica Sinica 49(3):404-407,2003].     

Use of alligator hole abundance and occupancy rate as indicators for restoration of a human-altered wetland

Use of indicator species as a measure of ecosystem conditions is an established science application in environmental management. Because of its role in shaping wetland systems, the American alligator (Alligator mississippiensis) is one of the ecological indicators for wetland restoration in south Florida, USA. We conducted landscape-level aerial surveys of alligator holes in two different habitats in a wetland where anthropogenic modification of surface hydrology has altered the natural system. Alligator holes were scarcer in an area where modified hydrology caused draining and frequent dry-downs compared to another area that maintains a functional wetland system. Lower abundance of alligator holes indicates lack of alligator activities, lower overall species diversity, and lack of dry-season aquatic refugia for other organisms. The occupancy rate of alligator holes was lower than the current restoration target for the Everglades, and was variable by size class with large size-class alligators predominantly occupying alligator holes. This may indicate unequal size-class distribution, different habitat selection by size classes, or possibly a lack of recruitment. Our study provides pre-restoration baseline information about one indicator species for the Everglades. Success of the restoration can be assessed via effective synthesis of information derived by collective research efforts on the entire suite of selected ecological indicators.     

An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia?×?hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounded shoot apical meristems induced the most effective and consistent silencing compared to other methods of inoculation. From an evaluation of ten cultivars, a compact petunia variety, 'Picobella Blue', exhibited a 1.8-fold higher CHS silencing efficiency in corollas. We determined that 20 °C day/18 °C night temperatures induced stronger gene silencing than 23 °C/18 °C or 26 °C/18 °C. The development of silencing was more pronounced in plants that were inoculated at 3–4 versus 5 weeks after sowing. While petunias inoculated with pTRV2-NbPDS or pTRV2-PhCHS showed very minimal viral symptoms, plants inoculated with the pTRV2 empty vector (often used as a control) were stunted and developed severe necrosis, which often led to plant death. Viral symptoms were eliminated by developing a control construct containing a fragment of the green fluorescent protein (pTRV2-sGFP). These optimization steps increased the area of chalcone synthase (CHS) silencing by 69 % and phytoene desaturase (PDS) silencing by 28 %. This improved VIGS protocol, including the use of the pTRV2-sGFP control plants, provides stronger down-regulation for high-throughput analyses of gene function in petunia.     

Allele Distributions and Frequencies of the Six Prion Protein Gene (PRNP) Polymorphisms in Asian Native Cattle, Japanese Breeds, and Mythun (Bos frontalis)

Six polymorphic sites of the bovine prion protein gene (PRNP) were genotyped in 569 animals of Asian native cattle, Japanese breeds, purebred mythun (Bos frontalis), and mythun × cattle composite animals. At the 23-bp indel site, a deletion (23?) allele was a major allele in all populations except mythun. At the 12-bp indel site, an insertion (12+) allele was a major allele in all populations. The 14-bp indel site was polymorphic in all Asian native cattle. In the octapeptide repeat region, a six-repeat allele was a major allele in all populations, and 5/5 and 4/6 genotypes were detected in Japanese Black and Mongolian cattle and in mythun, respectively. Two nonsynonymous single nucleotide polymorphisms (SNPs) (K3T and S154N) were detected in Asian native cattle and mythun. Haplotype analysis using the genotypes of the six sites estimated 33 different haplotypes. The haplotype 23? 12? K 6 S 14+ was found in all populations.     

Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator

Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.     

The visual system of the alligator

The eye tissues and liver of the alligator contain vitamin A₁ alone. The retina contains rhodopsin, typical in absorption spectrum (λ_max 500 mµ); but synthesized in solution from neo-b retinene and opsin much more rapidly than are the frog, mammalian, or chicken rhodopsins previously examined. In this regard alligator rhodopsin resembles the rhodopsins and porphyropsins of fishes, all of which so far investigated are synthesized rapidly in solution. The rates of synthesis in vitro of frog and alligator rhodopsins are matched closely by the rates of rod dark adaptation in living frogs and alligators, measured electrophysiologically at the same temperature. Alligator rods dark-adapt, and alligator rhodopsin is synthesized in solution, at rates characteristically associated with cones and cone pigments in frogs, mammals, and birds.     

Fluctuating water depths affect American alligator (Alligator mississippiensis) body condition in the Everglades,Florida, USA

Successful restoration of wetland ecosystems requires knowledge of wetland hydrologic patterns and an understanding of how those patterns affect wetland plant and animal populations. Within the Everglades, Florida, USA restoration, an applied science strategy including conceptual ecological models linking drivers to indicators is being used to organize current scientific understanding to support restoration efforts. A key driver of the ecosystem affecting the distribution and abundance of organisms is the timing, distribution, and volume of water flows that result in water depth patterns across the landscape. American alligators (Alligator mississippiensis) are one of the ecological indicators being used to assess Everglades restoration because they are a keystone species and integrate biological impacts of hydrological operations through all life stages. Alligator body condition (the relative fatness of an animal) is one of the metrics being used and targets have been set to allow us to track progress. We examined trends in alligator body condition using Fulton's K over a 15 year period (2000–2014) at seven different wetland areas within the Everglades ecosystem, assessed patterns and trends relative to restoration targets, and related those trends to hydrologic variables. We developed a series of 17 a priori hypotheses that we tested with an information theoretic approach to identify which hydrologic factors affect alligator body condition. Alligator body condition was highest throughout the Everglades during the early 2000s and is approximately 5–10% lower now (2014). Values have varied by year, area, and hydrology. Body condition was positively correlated with range in water depth and fall water depth. Our top model was the “Current” model and included variables that describe current year hydrology (spring depth, fall depth, hydroperiod, range, interaction of range and fall depth, interaction of range and hydroperiod). Across all models, interaction between range and fall water depth was the most important variable (relative weight of 1.0) followed by spring and fall water depths (0.99), range (0.96), hydroperiod (0.95) and interaction between range and hydroperiod (0.95). Our work provides additional evidence that restoring a greater range in annual water depths is important for improvement of alligator body condition and ecosystem function. This information can be incorporated into both planning and operations to assist in reaching Everglades restoration goals.