Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes.

BACKGROUND: Neurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development. METHODS: Here, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation. RESULTS: We observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4. CONCLUSIONS: Neurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.

Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli.

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-ß3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-ß3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-ß3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-ß3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-ß3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration.

Regulation of the tenogenic gene expression in equine tenocyte-derived induced pluripotent stem cells by mechanical loading and Mohawk.

Cell-based therapeutic strategies afford major potential advantages in the repair of injured tendons. Generation of induced pluripotent stem cells (iPSCs) expands cell sources for "regenerative" therapy. However, its application in tendon repair is still limited and the effects remain unclear. In this study, equine tenocyte-derived iPSCs (teno-iPSCs) were generated by expressing four Yamanaka factors. Compared to parental tenocytes and bone marrow derived mesenchymal stem cells (BMSCs), the transcriptional activities of lineage-specific genes, including Mkx, Col1A2, Col14, DCN, ELN, FMOD, and TNC, were highly repressed in the resulting teno-iPSCs. Exposure to cyclic uniaxial mechanical loading increased the expression of Scx, Egr1, Col1A2, DCN, and TNC in teno-iPSCs and the expression of Scx, Egr1, DCN, and TNC in BMSCs. Reintroduction of tenogenic transcription factor Mohawk (Mkx) upregulated the expression of DCN in teno-iPSCs and the expression of Scx, Col14, and FMOD in BMSCs. Mechanical loading combined with ectopic expression of equine Mkx further enhanced the expression of Egr1, Col1A2, DCN, and TNC in teno-iPSCs and the expression of Scx, Egr1, and TNC in BMSCs. These data suggest that the repressed lineage-specific genes in the teno-iPSCs can be re-activated by mechanical loading and ectopic expression of Mkx. Our findings offer new insights into the application of iPSCs for basic and clinic research in tendon repair.

Arsenic-induced sumoylation of Mus81 is involved in regulating genomic stability.

Chronic environmental exposure to metal toxicants such as chromium and arsenic is closely related to the development of several types of common cancers. Genetic and epigenetic studies in the past decade reveal that post-translational modifications of histones play a role in metal carcinogenesis. However, exact molecular mechanisms of metal carcinogenesis remain to be elucidated. In this study we found that As2O3, an environmental metal toxicant, upregulated overall modifications of many cellular proteins by SUMO2/3. Sumoylated proteins from arsenic-treated cells constitutively expressing His6-SUMO2 were pulled down by Ni-IDA resin under denaturing conditions. Mass spectrometric analysis revealed over 100 proteins that were potentially modified by sumoylation. Mus81, a DNA endonuclease involved in homologous recombination repair, was among the identified proteins whose sumoylation was increased after treatment with As2O3. We further showed that K10 and K524 were 2 lysine residues essential for Mus81 sumoylation. Moreover, we demonstrated that Mus81 sumoylation is important for normal mitotic chromosome congression and that cells expressing SUMO-resistant Mus81 mutants displayed compromised DNA damage responses after exposure to metal toxins such as Cr(VI) and arsenic.

Mps1 is SUMO-modified during the cell cycle.

Mps1 is a dual specificity protein kinase that regulates the spindle assembly checkpoint and mediates proper microtubule attachment to chromosomes during mitosis. However, the molecular mechanism that controls Mps1 protein level and its activity during the cell cycle remains unclear. Given that sumoylation plays an important role in mitotic progression, we investigated whether Mps1 was SUMO-modified and whether sumoylation affects its activity in mitosis. Our results showed that Mps1 was sumoylated in both asynchronized and mitotic cell populations. Mps1 was modified by both SUMO-1 and SUMO-2. Our further studies revealed that lysine residues including K71, K287, K367 and K471 were essential for Mps1 sumoylation. Sumoylation appeared to play a role in mediating kinetochore localization of Mps1, thus affecting normal mitotic progression. Furthermore, SUMO-resistant mutants of Mps1 interacted with BubR1 more efficiently than it did with the wild-type control. Combined, our results indicate that Mps1 is SUMO-modified that plays an essential role in regulating Mps1 functions during mitosis.

Sumoylation of Kif18A plays a role in regulating mitotic progression.

BACKGROUND: Kif18A, the kinesin-8 motor protein, plays an essential role in regulating alignment of bi-oriented chromosomes at the midzone during mitosis. Kinesin proteins, including Kif18A, are often deregulated in many types of cancers and are thought to play a critical role in cancer progression. However, little is known about the post-translational modifications of Kif18A and their effects on its biological activity. METHODS: Kif18A was identified to be a SUMO2 acceptor by using Ni-IDA resin to precipitate proteins from cells stably expressing His6-SUMO2. To identify the potential lysine residues, multi-site directed mutagenesis together with transient transfection and Ni-IDA pull-down assay were carried out. The confocal time-lapse imaging and immunofluorescent staining were used to study the roles of SUMO2 modification on Kif18A's activity during the cell cycle. RESULTS: Kif18A is covalently modified by SUMO2 during the cell cycle, and its sumoylation peaks at metaphase and then rapidly decreases upon anaphase onset. Mutational analysis identifies multiple lysine residues (K148, K442, K533, K660 and K683) as potential SUMO acceptors. The functional studies reveal that sumoylation of Kif18A has little effect on protein stability and subcellular localization. However, compared with the wild-type control, ectopic expression of SUMO-resistant mutants of Kif18A results in a significant delay of mitotic exit. Confocal microscopy shows that cells expressing SUMO-resistant Kif18A display a compromised dissociation of BubR1 from kinetochores after anaphase onset. CONCLUSIONS: Our studies reveal that sumoylation functions as an unidentified form of post-translational modification that regulates Kif18A activity during mitotic progression.

Identification of the nuclear localization signal of SALL4B, a stem cell transcription factor.

SALL4B plays a critical role in maintaining the pluripotency of embryonic stem cells and hematopoietic stem cells. SALL4B primarily functions as a transcription factor, and, thus, its nuclear localization is paramount to its biological activities. To understand the structural basis by which SALL4B was transported and retained in the nucleus, we made a series of SALL4B constructs with deletions or point mutations. We found that K64R mutation resulted in a random distribution of SALL4B within the cell. An analysis of neighboring amino acid sequences revealed that (64)KRLR (67) in SALL4B matches exactly with the canonical nuclear localization signal (K-K/R-x-K/R). SALL4B fragment (a.a. 50-109) that contained KRLR was sufficient for targeting GFP-tagged SALL4B to the nucleus, whereas K64R mutation led to a random distribution of GFP-SALL4B signal within the cell. We further demonstrated that the nuclear localization was essential for transactivating luciferase reporter gene driven by OCT4 promoter, a known transcriptional target of SALL4B. Therefore, our study identifies the KRLR sequence as a bona fide nuclear localization signal for SALL4B.

Nuclear protein IK undergoes dynamic subcellular translocation and forms unique nuclear bodies during the cell cycle.

IK is a nuclear protein containing a unique domain named RED due to the presence of a repetitive arginine (R), aspartic (E), and glutamic acid (D) sequence. To date, the function of this protein remains largely unknown despite of a couple of previous studies in the literature. Here we report that depletion of IK via RNA interference results in mitotic arrest. We also demonstrate that IK undergoes dynamic translocation during interphase and mitosis. In particular, IK is primarily present in some interphase cells as nuclear foci/bodies which do not co-localize with nucleoli, PMA bodies and Cajal bodies. Pull-down analysis coupled with mass spectrometry reveals that IK is associated with DHX15, a putative ATP-dependent RNA helicase. Our results strongly suggest that IK may participate in pre-mRNA splicing and that it may be a useful biomarker for a new nuclear structure in the cell.

Sumoylation is important for stability, subcellular localization, and transcriptional activity of SALL4, an essential stem cell transcription factor.

SALL4 is a transcription factor that plays a key role in the maintenance and self-renewal of embryonic stem cells and hematopoietic stem cells. Given that little is known about regulation of SALL4, we studied biochemical modifications of SALL4B, a major splicing variant of SALL4, and elucidated their biological function. SALL4B was primarily modified by ubiquitination when it was expressed in both Sf9 and HEK293T cells. A significant fraction of SALL4B was further modified by sumoylation when it was expressed in HEK293T cells. Constitutive SUMO-modification of SALL4B was also detected in Tera-1, a cell line of the teratocarcinoma origin. SALL4B sumoylation was independent of ubiquitination and lysine residues 156, 316, 374, and 401 were essential for sumoylation. Chromatin fraction contained more SUMO-deficient SALL4B. Despite a shorter half-life than the wild-type counterpart, SUMO-deficient SALL4B interacted with OCT4 more efficiently than the wild-type SALL4B. RNAi-mediated silencing of SALL4 expression caused significant down-regulation of both OCT4 and SOX2, which was rescued by ectopic expression of SALL4B but not by SUMO-deficient mutant. Significantly, compared with the wild-type SALL4B, SUMO-deficient mutant exhibited compromised trans-activation or trans-repression activities in reporter gene assays. Combined, our studies reveal sumoylation as a novel form of post-translational modification for regulating the stability, subcellular localization, and transcriptional activity of SALL4.

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing.

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.

BubR1 is modified by sumoylation during mitotic progression.

BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.

Chromium induces chromosomal instability, which is partly due to deregulation of BubR1 and Emi1, two APC/C inhibitors.

Disruption of cell cycle checkpoints and interference with the normal cell cycle progression frequently result in cell death or malignant transformation. Hexavalent chromium [Cr(VI)] is a well-known carcinogen that has been implicated in the occurrence of many types of human malignancies, including lung cancer. However, the exact mechanism by which Cr(VI) causes malignant transformation in the lung remains unknown. We have demonstrated that chronic exposure to a non-cytotoxic concentration of Cr(VI) induced a variety of chromosomal abnormalities, including premature sister chromatid separation, chromosomal breakage and the presence of lagging/misaligned chromosomes. After treatment with nocodazole, both HeLa and normal lung bronchial epithelial cells were arrested at mitosis. However, Cr(VI) significantly compromised M-phase arrest induced by nocodazole. Cr(VI) suppressed BubR1 activation and reduced expression of Emi1, leading to an unscheduled activation of APC/C. Consistent with this observation, Cr(VI) treatment caused enhanced polyubiquitination of geminin during mitotic release, while it deregulated the activity of Cdt1, a DNA replication licensing factor. Combined, these results suggest that Cr(VI)-induced chromosomal instability is partly due to a perturbation of APC/C activities, leading to chromosomal instability.

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in poly (ADP-ribose) polymerase (Parp-1) null oocytes.

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1((-/-)) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1((-/-)) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.

Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes.

BACKGROUND: In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. RESULTS: When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. CONCLUSION: Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species.

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

A preliminary study on the rheological properties of human ejaculate and changes during liquefaction.

AIM: To study the changes in rheological properties, namely the parameters of the hysteresis loops and yield stress versus time for human semen after ejaculation. METHODS: Ejaculates were obtained from volunteers and immediately put into the test cup of a Brookfield Programmable DV-11 Rheometer, by which the hysteresis loops and yield stress were determined. RESULTS: (1) Yield stress values dropped down from more than 3000 mPa to 60 mPa in about 5 minutes after ejaculation; (2) The shape of the hysteresis loops of shear stress versus shear rate was changed from the counter-clockwise direction, that enclosed a large area, into the clockwise direction, that enclosed a very small area. CONCLUSION: Human ejaculate originally possesses semi-solid or visco-elastic body behavior and in 5 minutes after liquefaction, it becomes a thixotropic fluid or shearing thinning fluid with very low viscosity.

[Molecular cloning and analysis of bovine prolactin full-long genomic as well as cDNA sequences].

In this report, a full-length sequence of bovine prolactin (bPRL) genomic DNA with 9388 bp, which has been accepted by GenBank (Accession Number: AF426315), was firstly cloned by Long PCR procedures. This sequence consists of 5 exons, 4 introns, 854 bp of 5' upstream regulatory region and 69 bp of 3' UTR. Accession number of protein encoded by AF426315 sequence in GenBank is AAL28075 that is composed of 229 amino acid residues, in which signal sequence resides in 1-30 sites and mature polypeptide consists of 199 amino acid residues. The recombinant plasmid containing bPRL genomic DNA was then transfected into eukaryotic cells (COS-7), followed by RT-PCR procedure. An 804 bp of bPRL cDNA containing all the encoding region was obtained, indicating that the bPRL genomic DNA reported herein had its biological function at the transcriptional level. Results derived from information searching by Blast program revealed that there were various SNP sites in the sequences of bPRL mRNAs and ESTs collected in GenBank, which located mainly in downstream encoding region and 3' UTR. These SNP sites did not alter the related amino acids encoded. In addition, mRNA sequences encoding 5' signal sequence of bPRL gene was highly conserve.