Objective: To study the espression of pepsinogen C gene in epithelial cells in developing rat stomachs. Methods: In this study, the ontogeney of pepsinogen producing cells in rat fundic glands was studied by in situ hybridization vsing a digoxigenin-labeled RNA probe. Results: The rat gastric epithelium was stratified but was morphologically undifferentiated at 18.5 days gestation. The pepsinogen mRNA was expressed both in chief cells and mucous neck cells in adult rats, in which first detected by in situ hybridization in the stomach of the rats at 3.5 days after birth. The development of pepsinogen producing cells could be classified into for stages:①18.5 days gestation to 0.5 days after birth;②3.5 days to 2 weeks atrer birth;③3-4 weeks after birth; ④8 weeks after birth. Pepsinogen expression is strictly limited to these cells and shown a developmetal stage-specific manner. Conclusion: We concluded the pepsinogen C could offer excellent molecular markers of differentiation of stomach epithelial cells in developmental biology.
Objective The purpose of this paper is to study the effect of estradiol on testosterone secretion in cultured Leydig cells of male rhesus monkey in vitro. Method Testes were dispersed and mixed with 0.1% collagense and loaded onto BSA density gradients. The cultured media were collected for testosterone RIA. Result The results showed that pregnenolone, progesterone, 17α hydroxypregnenolone enhanced the stimulatory testosterone secretion, the amount of testosterone secretion increased 1 2-5 6 fold more than the control. The higher concentration appeared on the 17α hydroxypregnenolone (2.3-5.6 fold more than the control). It was found that estradiol decreased the stimulatory action of pregnenolone, progesterone, 17α hydroxypregnenolone on testosterone secretion. Conclusion According to correlative analyzed results, it is suggested that effect of estradiol on testosterone secretion in Leydig cells of male rhesus monkey could be due to the inhibitory effect of 3β delydrosterolase, 17α hydroxylase and 17,20 desmolase activity.
Plant genetic transformation is already sufficiently developed to allow the testing and even commercialization of plants with novel phenotypes under simple genetic control, and it is now a core research tool in plant biology and a practical tool for cultivar and tree improvement. Although plant genetic transformation systems using Agrobacterium and particle bombardment are comprehensively addressed, the major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review will discuss the biological and practical requirements for transformation systems; the strategies to achieve gene transfer and transgene expression, verification of plant genetic transformation, and transgenic plants and public perceptions.
Producing of pharmaceutical proteins in milk of transgenic animals is one of the most promising areas in modern biotechnology. To establish transgenic mice model of mammary gland bioreactor for production of human recombinant thrombopoietin, the mammary gland-specific expression vector pWAPTPO was constructed using 5' regulatory sequence of whey acidic protein (mWAP) gene and 3' control region of bovine ct-S1-casein gene. The chimeric gene was injected into mouse zygotes by regular pronuclear microinjection. 16 Go generation founder mice were obtained. The integration analysis indicates that 6 of them were transgenic, thus the rate of integration was 37.5% (6/16). The presence of recombinant human thrombopoietin was detected in the milk of transgenic mice at levels of up to 0.8 μg/mL. These results showed that the established transgenic mouse model could produce recombinant proteins in the milk of lactating females.
It is well known that modern Aves lack dentition, unlike their toothed ancestors of the Jurassic and cretaceous periods. However, in the chick oral cavity a rudiment forms that resembles the lamina stage of the mammalian molar tooth germ. We have previously addressed the molecular basis for this secondary loss of tooth formation in Aves by analyzing inchick embryos the status of molecular pathways known to regulate mouse tooth development.in this study, we further examined the molecular pathways involved in positioning the site of tooth rudiment. Similar to the mouse tooth germ, Pax9 serves as a marker for the presumptive dental mesenchyme in the chick prior to any morphological manifestation of odontogenesis. The restricted Pax9 expression in the chick mandibular mesenchyme is positioned by the tvo signals from overlying oral epithelium. FGF8 induces Pax9 expression while BMP4 prevents this induction. Thus it appears that, similar to that in mice, the position of tooth rudiment in chick embryo is determined by two different types of signaling molecules with antagonistic activity.These results further demonstrate that the early signaling pathways involved in odontogenesis remain inducible in Aves.
To isolate some differentially expressed genes during mouse spermatogenesis,mRNA differential display method was used in the present study to examine the differential genes expressed in day-60 mouse testis and round spermatids with day-16 testis and pachytene spermatocytes as a control, respectively. Totally, 12 fragments were cloned and sequenced. It is found by homology search that 5 of them (sp3, sp4, sp7, sp9 and sp10) were respectively well matched with known genes, 6 fragments displayed high homology with some cDNA or ESTs whose function were unknown, another fragment (sp12) showed no homology with known sequences. Northern blot hybridization analysis revealed that sp1 and sp8 were predominantly expressed in adult mouse testis. The full-length eDNA of sp1 was identified by 5'RACE. This gene encodes a putative highly phosphrylated protein of 541 amino acids containing a nuclear localization signals. No significant homologies with known genes were observed at the nucleotide and amino acids level. Limited amino acid homology with 2 protein domains was noted. Northern blot hybridization show enhanced expression in 20～60 day old testis. Tissue expression pattern appears to be highly restricted in testis. Considering their expression pattern, the possible involvement of this gene in meiotic and postmeiotic processes has been suggested. Based on these observations, we propose that we have identified a new gene, which we call peat (predominantly expressed in adult mouse testis).
The SOX genes of Panthera tigris altaica Temminck were detected by a pair of degenerate primers, which were special to the SOX gene family within the HMG-box. With the genomic DNA as template, one specific band was observed with the length of 220 bp. Five fragments of DNA were found and their identities to the SRY of human were 75%, 56%, 51%, 67% and 48%,respectively; to the Sty of mouse were 73%, 54%, 57%, 66% and 48%, respectively. The genes with the maximum similarity to these five fragments were H-SOX3, H-SOX4, H-SOX11, H-SOX21 and H-SOX22. So, these five DNA fragments are part of Sox genes of Panthera tigris altaica, and were named as PtSox3, PtSox4, PtSox11, PtSox21 and PtSox22 according to the general nomenclature.
The enzyme 3β-Hydroxysteroid dehydrogenase/△4-△5 isomerase (3β-HSD) is a key enzyme in the biosynthesis of steroid hormones. We have isolated cDNA encoding goat fetal 3β-HSD from a liver λgt10 cDNA library by RT-PCR and screening of the library. The 3′ end of the cDNA is deficient. RT-PCR products from total RNA of different liver show that the expression of 3β-HSD mRNA is alternative.
The gene Dad-1 is a well-conserved programmed cell death (PCD) suppressor in animals and plants. Using fluorescence in situ hybridization (FISH), the authors physically mapped the single copy rice Dad-1 gene onto the distal region of short arm in rice chromosome 2 for the first time. The homology of it in maize genome was also analyzed. The results of Southern blot suggested that there are sequences homologous to rice Dad-1 in maize genome. FISH further showed that the three hybridization signals were detected in maize, two of them on the long arms of chromosome 4 and 5, another on the short arm of chromosome 9, respectively. And the distances from the signal to centromere (FL value) were 91%, 98% and 96%, respectively. All of the three hybridization sites of maize were similar to those of rice in their relative position, they all located at the terminal regions of the chromosome arms. It indicated that, in a certain degree, gene Dad-1 was conservative not only for their homology of sequence but also for their positions on chromosome in rice and maize. The detection rate of the hybridization signals on rice chromosomes was higher than that in maize. It indicated that the Dad-1 coding sequence of rice was more easily to be hybridized with rice than that with maize chromosomes and it only meant the partial homology to that of maize.
In order to investigate the changes of circumstance of fertilization at the time pre- and post-ovulation, the amino acid content of cervix mucus in 27 cows were determined separately. The results indicated that the amino acid content at the time pre-ovulation in average was 1032 μg/mL, and 2380 μg/mL at the time post ovulation which made up 2.3 times of the former. The highest proportions of determined amino acids in cervix mucus were those of glutamic acid and threonine making up 12.8% and 12.2% of the total, respectively, and the lowest was that of histidine, making up only 2.2% of the total. Those proportions stand stable at the time pre- and post-ovulation. The threonine content in cervix mucus of the tested sample cows at the time post-ovulation dropped on an average of 19% of that at pre-ovulation. And, lysine increased at post-ovulation on an average 25% of that at pre-ovulation. The amino acid metabolism in body fluid is an important physiological activity, in cervix mucus after ovulation the both cases of dropping of threonine content and the enhancement of lysine reflect the changes in protein composition which were related to those amino acids. As for the aforesaid changes, it still needs further research on the relating physiological significance and the producing influence upon the combination between egg and X or Y sperm.
The method of dual pronuclear co-injection into fertilized egg was put forward.Identification of integration of exogenous genes from the pronuclei of 8-celled phase which were controlled under the regulation sequence of human granulocyte colony-stimulating factor (G-CSF) by using co-injection of two fragments of whey acidic protein (WAP) which have part of homologous domain, was conducted through PCR and Southern blotting techniques. The results indicated that the integration efficiency resulting from dual pronuclear injection revealed an increase by 62.65%,comparing to that from single injection, 48.68%. It provides a new pathway for raising integration efficiency in setting up transgenic animals.
By precisely controlling exogenous hormones applied in the cultural process,particular type of floral organs, such as tapels, stamens, or ovules, can be regenerated from perianth explant of Hyacinthus orientalis L. To further study the molecular mechanism of the specific organgenesis in the system, an AP2 homolog, HAP2 was isolated from regenerated tepals by using RT-PCR. Results from RT-PCR combined with Southern hybridization showed that H4P2 was expressed in leaves, perianth, regenerated tepals and regenerated stamens. The possible functions of HAP2 on hormone-regulated floral organ regeneration are discussed.
The changes of ADAMs and the variation of acid, neutral and alkaline proteinases in growth and development of rat liver were analyzed by SDS-PAGE, SDS-G-PAGE and Western blot.These results showed that the two activities peak, 30 kD and 32 kD acid proteinases, appear respectively in the liver of beginning suckling and the 5th-week-old rats. The activities of the 75/80 kD acid proteinases appeared only in the 3rd-week-old rat liver. The activities of the 71 and 75 kD neutral proteinases were at the highest in the 3rd-week-old rat liver. The 15 kD alkaline proteinase activity appeared in the beginning suckling rat liver. The 75 kD ADAMs was detected respectively in rat liver during the 20-day rat embryo to the 21-day-old, and during the 30-day-old to the adult body. A 40 kD ADAM was mainly detected in the rat embryo liver and in the suckling at liver, but a 30 kD ADAM mainly appeared in the feeding rat liver. The changes of the 50/47, 73 and 140 kD MDC15 were similar to the 75 kD ADAMs. A 30 kD MDC15 only in the 15～35th-day-old rat liver and a 58 kD MDC15 mainly appeared in the 14～49th-day-old rat liver. These results indicates that the nutritive source and form are able to effectively regulate the physiology and biochemistry of the rat liver, and that the function, growth and development of the rat liver have a close relationship with its nutrition.
In order to clarify the phenomenon of genetic variation in 4 different disease symptoms of apple disease, caused by Physalosporepiriola, at molecular level, and the degree of genetic variation in 4 isolates of pathogenetic bacteria, the DNA fingerprint analysis of 26 related bacterial isolates of Ph.piricola from three major productive regions of apple in China was made by using RAPD technique.The results indicated that (1) Obvious genetic differences at the level of genomic DNA were found in different pathogenic bacterial isolate, and fingerprint of 4 of 11 polymorphic primers of amplification could distinguish the above mentioned 4 bacterial isolates from each other, among which the genetic distance was revealed by 0.1314～0.2541 on the basis of calculation of analytic data; (2) High genetic similarity (88.4～100%) between the identical symptom types from different sources of pathogenic bacteria were shown. It thus indicates the insignificant relation between the genetic differentiation and geographic difference. Above results of research are of importance in theoretic demonstration about the heredity and evolution of the disease Ph. piricola in apple fruit as weU as in practice in improvement of identification and control technique for the disease.
Dad1 is a highly conserved programmed cell death (PCD) suppressor gene. Using digoxigenin-labeled anti-sense RNA probe, the expression of Dad1 cDNA in 4rabidopsis was detected in developing maize seed tissues, including ovary, endosperm and embryo. Our results of in situ hybridization revealed that Dad1 was expressed in degenerating tissues, such as the ovary wall, the integument (Fig.1 B&C), the mature endosperm (Fig.1 E) and the root cap (Fig.2 A), as well as in some vigorously metabolizing cells such as the meristematic cells of root tip (Fig.2 A&B), the cotyledon cells (Fig.2 C&D), the female germ cells (Fig.1 C) and the dividing endosperm cells (Fig. 1 C) in a temporal and spatial manner. These results show that, as other Dad1 homologs, maize Dad1 may also play a role in maize embryo development and in PCD during endosperm degeneration.
Populus tomentosa, as an important economic plant and as a natural host of Agrobacteriun tumefaciens, has been used as a model plant for tissue culture and genetic transformation study of xyloghyta. In this work, several important factors influencing the efficiency of Agrobacterium-mediated transformation and regeneration of plants, such as types of explants, concentration of Agrobacterium suspension, wounding treatment on explants, conditions of pre-cultivation and co-cultivation, medium composition etc, were studied, and an Agrobacterium-mediated high frequency system was established for P.tomentosa@ After pre-cultivation in the medium MSy for 48 h, the healthy sterile leaves were cut into discs, co-cultivated with suspension of A. tumefaciens LBA4404 at the concentration of 5x108 cells/mL for another 48 h, and then transferred onto the selective media containing appropriate antibiotics. The average transformation frequency of kanamysin resistant (Kmr)calli was up to 56.2%, and the ratio of the number of transgenetic plants to the number of explants was up to 19.9%. Only by wounding treatment alone, the transformation frequency was improved by 20～40%. With this transformation system, NP-1, a disease resistant gene, was successfully introduced into P. tomentosa and a lot of transgenetic plants were obtained.
Using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and immunohistochemical methods, the pollination-induced cell death in tobacco (Nicotiana tabacum L.) was detected. The results indicated that pollination induced apoptotic death in specific cells. Apoptosis was initiated during the tetrad stage after meiosis in the tapetal cells, cyclic cells, and connective cells (Plate I, C and D), and during pollen mother cell stage in the vascular bundle (Plate I, E). Those apoptotic events, which occurred in the specific cells in the specific stages of anther development, play some vital roles of specific biological function. The highly expression of calcium/calmodulin-dependent protein kinase T1 related closely to these apoptotic processes (Plate II) in these cells, suggesting that calcium-mediated signal pathway is involved in the regulation of apoptosis during anther development in tobacco.
In the development of techniques by using a bioreactor to produce a special protein,and the establishment of a temporary expression system from primary cell culture of special tissue to analyze the expression regulators and the recombinant gene will greatly expedite the progress of research. This paper is the first report on the establishment of a temporary expression system from the primary culture of chicken oviduct epithelium, which continuously synthesized and secreted ovalbumin (OA) during more than two weeks of incubation.When the secretion function of the cells decreased, it could be restored in most of the cells by adding hormones in the culture medium within one week of incubation. The OA can be quickly and easily measured by using dot immuofiltration assay in the cell culture medium because it is a kind of secretary protein. In order to examine whether the exogenous genes can be expressed in the primary culture cells, the green fluorescent protein gene (GFPG) was transfected into oviduct cells, and green fluorescence was observed in the cytoplasm of the epithelial cells. These results indicate that the temporary expression system in chicken oviduct epithelium is an effective and convenient system to analyze the expression regulators and the recombinant gene.
Leukemia inhibitory factor (LIF) is essential for embryo implantation andmay mediate the roles of ovarian estrogen and progesterone during embryo implantation.LIF can also cooperate with other factors, such as Homebox gene, heparin-binding EGF-likegrowth factor and cyclooxyge nase-2, and mediate or regulate the roles of these moleculesduring embryo implantation.
