Construction Of Neural Tube CDNA Library And Screeniong Of Genes Associated With NTDs Caused By Hyperthermia

Neurulation ~ from the appearance of the neural plate to the closure of the neural groove ~ is a fundamental event in embryogenesis and the first step in development of the central nervous system. During this process, should the neural tube fail to close accurately and at the proper time, the result is a neural tube defects (NTDs). The resulting malformation can manifest itself in many ways, e.g., encephalomyelocele, anencephaly, or exencephaly. The frequency of NTDs is about 1 per 1000 pregnancies, making them the commonest sort of congenital malformation. Although the morphological events comprising neurulation have been described in detail, the underlying molecular mechanism of neural tube closure remains poorly understood.NTDs result from both genetic and environmental factors. Among the latter causes, hyperthermia is a common physical teratogen, as demonstrated by both epidemiological studies and animal experiments. Embryonic development at a few degrees above normal body temperature frequently causes congenital malformations, the commonest among these being NTDs. This observation has motivated extensive research into the mechanism by which hyperthermia causes NTDs. There is now an extensive literature on the effects of hyperthermia on cellproliferation, differentiation, migration, adhesion, aggregation, and apoptosis, as well as on tissue induction and organogenesis. Genes have been identified whose excessive or insufficent expression induces neural tube malformation with resulting NTDs. However, the current understanding is only beginning to explain the complex and orderly sequence of events comprising neurulation. To further investigate the many genes that contribute to neurulation and to determine how their dysfunction leads to NTDs, we have constructed cDNA libraries from neural tubes taken from hamster embryos.We constructed a cDNA libraries using neural tubes removed from E8.5 embryos of golden hamster, that being the developmental stage when neurulation is in progress. It was confirmed that the cDNA libraries were complete and consisted of full-length transcripts. Those facts being established, PCR amplification according to the subsection screening method has been used to isolate two genes that are related to the development of the neural tube. Thanks to their sequence homology to characterized genes from rats and mice, the two hamster sequences have been identified as RPL30 and HMGB1. Their responsiveness to the ambient temperature combined with their expression in the neural tube implicates them in its developmental malformation at elevated temperatures. Both cDNA segments have been digoxin-labeled, enabling us to measure the levels of RPL30 and HMGB1 transcripts in tissue samples. Using the method of northern blotting, the temporal pattern of gene expression has been determined for both species throughout the development of the neural tube and NTDs.PART 1: CONSTRUCTION AND CHARACTERIZATION OF cDNA LIBRARY FROM NEURAL TUBE OF GOLDEN HAMSTERIn order to discuss the mechnism underlying neurulation and NTDs, we constructed a neural tube cDNA library of gloden hamster at neurulation firstly.Because genomic DNA are very complex, and have abundant repeated sequences, it is difficult to obtain target gene directly. While cDNA library is simple to screen. Therefore, constructing a cDNA library is the basic method on the molecular biological research. Now, We use the SMART method to construct a cDNA library with high quality. Total RNAs were isolated from the neural tube of E8.5d golden hamster using TRIZOL Reagent; First-stranded cDNAs were synthesized using SMART IV Oligonucleotide and CDSIII/3' PCR Primer as primers; Double-stranded cDNAs were synthesized using LD-PCR method; The dscDNAs were digested by Proteinase K and Sfi I(IA & IB) enzyme, less than 500 bp fragments were separated by CHROMA SPIN-400 column to ensure the length of inserts, then the cDNAs were ligated to the Sfi I A & B-digested A TriplEx2 vector. After packaging, the neural tube cDNA library was constructed. The titer of the primary library was 1.5 X 106pfu/ml, the recombination rat was 99%, the average insert was larger than lkb, the liter of the amplified neural tube cDNA library was 1 X 109pfu/ml, and the P -actin gene segment of golden hamster was amplified from the constructed cDNA library. Thus, Our successfully constructed cDNA library was a full-length library with high efficiency.PART 2: CLONING OF GOLDEN HAMSTER RPL30 GENE AND STUDIES ON THE RELATIONSHIP BETWEEN RPL30 AND THE DEVELOPMENT OF THE NEURAL TUBE ANDNTDs CAUSED BY HYPERTHERMIAIt is not our final purpose to construct a cDNA library but to use it to identify some interesting target genes. Our aim is to screen some key genes associated with the development of the neural tube and NTDs caused by hyperthermia from this neural tube cDNA library that we constructed.There are many methods for screening genes from library such as nucleic acid hybridization and immune screening, but each has advantages and disadvantages.Now, we select SSS method to subsection screen target genes from the cDNA library. With this method, cDNA phage plate was divided into several blocks and target genes were screened from the library by PCR method. Comparing with other methods, this method has many advantages such as controlled range and clear target, and also quick and simple for obtaining the target gene.Factors that affect the neurulation process come from inside and outside of the neural tube, many are some conservative genes regulating morphogenesis or some oncogenes. Now, we choose ribosomal proteins as target genes. Ribosomal proteins assemble ribosome with ribosomal RNA, and involve in protein biosynthesis and translation. The expression of ribosomal proteins may reflect the growth state of cells. Ribosomal protein express low or not may involve in some pathological processes, and some ribosomal protein genes may be oncogenes. RPL 30 (ribosomal protein Z30)gene is one of the abundant RP genes, and belongs to the so-called housekeeping gene category, composing 60S ribosomal subunits, maintaining cell growth and survival. RPL30 gene is similar to RPL32 which has secondary functions for morphogen. Hence, a couple of primers was designed according to RPL30mRNA sequence of mouse. First, the limit dilution of the library containing a positive clone was ensured to be 10"2, and the suitable PCR condition was built, then the primary positive signal pool> the secondary positive signal pool and the single target plaque were obtained by screening the library with PCR method step by step. The nucleotide sequences of cDNA inserts was determined and analyzed with BLAST, results show that the length of the cDNA insert is 535bp, containing full ORF of 348bp (residuesl 12-459) , and had high homologous to the gene of mouse and rat. The amino acid sequences was 115 of RPL30 protein, with high homologus to other species. This results arrived at our project, and we obtained RPL30mKNA sequences of golden hamster.There is no report about the role of RPL30 gene in the development of the neural tube and NTDs caused by hyperthermia. But someone reported that a temperature shift can cause immediate inhibition of the transcription and the translation of all ribosomal proteins. In order to discuss the relationship between- Q -RPL30 gene and the development of the neural tube and NTDs caused by hyperthermia, the RPL30cDNA was extracted > purified and labelled with digoxigenin firstly. Total RNAs were isolated from the neural tube at each time during neurulation and NTDs group, the level of RPL30mRNA in the neural tube was detected by Northern blot during the neural tube development and NTDs caused by hyperthermia. We have discovered that the level of RPL30mRNA was high in the neural tube at each time, increased gradually during neurulation, and increased significantly at E12d group, whereas decreased at the H8.5d NTDs group. It has been suggested that in response to hyperthermia, the transcription of RPL30mRNA had been severely inhibited, all ribosomal genes relevant to RPL30 gene was also inhibited, as a consequence several protein biosynthesis was reduced or stopped, some of which invloved in neual tube development. Thus this will inhibit neural tube closure, and may cause NTDs.PART 3: CLONING OF GOLDEN HAMSTER HMGB1 GENE AND STUDIES ON THE RELATIONSHIP BETWEEN HMGB1 AND THE DEVELOPMENT OF THE NEURAL TUBE AND NTDs CAUSED BY HYPERTHERMIADuring PCR screening of recombined cDNAs from the constructed library, we obtained a recombined plaque that appeared frequently. The cDNA insert was detected to be 1.6Kb long. After being converted to plasmid, enzyme identified and sequence analysis, the cDNA insert was confirmed to be 1242bp, containing full ORF, with start codon ATG (residues 97-99) and termination codon TAA (residues 742-744), and displayed high homologous to the HMGBl gene of other species; the amino acid sequences was 215 of HMGBl protein, with high homologus to other species. Thus this insert cDNA was a full-length cDNA of HMGBl. But there was no recording about HMGBJmRNA of golden hamster in Genebank, now we obtained this message.HMGBl is a nonhistone protein componets of chromatin, and is an extremely well conservative protein across speices. In rat and mouse brains, expressions of HMGBl are high during the embryonic period, HMGBl has been suggested to be involved in neurite outgrowth, neuron differentiation and neural crest cell migration during development. Significantly, HMGB1-SGC interaction is important for cell-cell recognition and cell migration. SGC (Sulfoglucuronyl Cargbohydrate ) is expressed in several glycolipids -. glycoproteins and proteoglycans of the nervous system, several cell adhesion molecules such as NCAM express SGC, and neural crest cell also express SGC. SGC has been implicated in migration of cell-cell recognition and neural crest cell migration. As we know, neural crest cell begin migration before neural tube closure. In the cranial region, neural crest cells migrate from the neural folds early in the process of elevation, which is necessary for neural tube closure. However, less is known about the relationship between HMGBl gene and the development of the neural tube and NTDs caused by hyperthermia.In order to discuss the role of HMGBl gene in the development of the neural tube and NTDs caused by hyperthermia, the HMGBlcDNA was extracted > purified and labelled with digoxigenin. Total RNAs were isolated from the neural tube at each time and NTDs group. The level of HMGBlmRNA in the neural tube was detected by Northern blot during the development of the neural tube and NTDs caused by hyperthermia. Northern analysis showed that the level of HMGBlmRNA was increased gradually during neurulation, and increased significantly at ElOd, and also high in the neural tube at ElldN E12d, whereas decreased at the H8.5 NTDs group. It has been suggested that hyperthermia made the transcription of HMGBlmRNA decline, induced HMGBl protein biosynthesis, affected HMGBl-SGC interaction, inhibited neural crest cell migration, and as a result the neural folds can not fuse in the midline and the neural tube failed to close, resulting in NTDs. Thus, HMGBl gene is necessary for the neural tube development and differentiation. It seems likely that after hyperthermia, HMGBl gene express low may have a crucial role in NTDs caused by hyperthermia.___ -11In this study, we constructed a neural tube cDNA library of gloden hamster with full-length and high effencicy. On the basis of this, we firstly subsection screened two genes(RPL30 and HMGBl) associated with the neural tube development by SSS methods, then discussed the relationship between RPL30^ HMGBl and the development of the neural tube and NTDs caused by hyperthermia using Northern blot method.