Age-Dependent Organotypic Expression of Microtubule-Associated Proteins (MAP1, MAP2, and MAP5) in Rat Brain

Age-dependent changes in the distribution of microtubule-associated proteins (MAPs) were analyzed in young (3-months, N = 3) and old (24-months, N = 3) rat brain. In the young rats, MAP1 and MAP5 exhibited prominent immunostaining in the perikarya and dendrites whereas MAP2 was selectively localized in the dendrites. In the cerebellum, MAP2 was preferentially localized in finer and distal branches of Purkinje cell dendrites and in punctate deposits surrounding glomeruli. In general, aging resulted in obvious declines in MAP2- >> MAP1- and MAP5-immunoreactivities in the hippocampus and parietal cortex but no change in cerebellum. The results indicate that: (1) hippocampus is the most affected and cerebellum is the least affected region with regard to declines in MAPs-immunoreactivities in the aged rat brain; (2) dendrite-specific MAP2 is almost completely depleted from most dendrites in the hippocampus and cortex. In summary, loss of MAP2-immunoreactivity in the affected brain areas may be associated with age-related impairment of synaptic plasticity, cognition and memory functions.     

Organization, Sequence, and Expression of a Gene Encoding Goldfish Neurofilament Medium Protein

Abstract: The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF-M) protein. The tissue-specific expression of goldfish NF-M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF-M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF-M protein is similar to higher vertebrate NF-M proteins. In addition, the organization of the goldfish NF-M gene is identical to the NF-M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF-M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF-M genes.     

Organization of the Human Protein 4.1 Genomic Locus: New Insights into the Tissue-Specific Alternative Splicing of the Pre-mRNA

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3′-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3′ UTR, over 3 kb, accounts for one-half of the mature mRNA.     

Genomic Organization,Fine-Mapping,and Expression of the HumanIslet-Brain 1 (IB1)/C-Jun-Amino-Terminal Kinase Interacting Protein-1 (JIP-1)Gene

Islet-brain 1 (IB1), a regulator of the pancreatic β-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the humanIB1gene, and the characterization of anIB1pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. TheIB1gene contains 12 exons and maps to chromosome 11 (11p11.2–p12), a region that is deleted in DEFECT-11 syndrome. Apart from anIB1pseudogene on chromosome 17 (17q21), no additionalIB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.     

Tissue-Specific Expression of the Gene Encoding a Mouse RNA Binding Protein Homologous to Human HuD Antigen

We have cloned and sequenced cDNAs encoding a mouse RNA-bindingprotein that is homologous to human HuD antigen. The amino acidsequence deduced from the nucleotide sequence has revealed thatthe mouse HuD protein is identicalto the human counterpart exceptfor two amino-acid substitutions outside the three RNA recognitionmotifs (RRMs) and a difference in the N-terminus. The mouseHuD gene produces two major brain-specific mRNAs (3.7 kb and4.4 kb) and a minor testis-specific mRNA (1.3 kb), which isindicative of alternative RNA processing. These results suggestthat the mouse HuD homolog is a member of the tissue-specificRNA-binding protein family, possibly involved in RNA metabolismin the nervous system.     

人促红细胞生成素基因组基因和cDNA的克隆及其在COS－7细胞中的表达

利用ＰＣＲ技术,从正常人胎肝染色体ＤＮＡ中克隆到长度为１５７２ｂｐ的人促红细胞生成素（ＥＰＯ）基因组基因片段,它包含除第一个外显子和第一个内含子外所有外显子及内含子。再人工合成１３ｂｐ外显子１的编码区,并与１５７２ｂｐ片段拼接,从而得到除第一个内含子的人促红细胞生成素基因组基因。将克隆得到的ＥＰＯ基因插入载体ｐＳＶ２－ｄｈｆｒ得到ｐＳＶ２－ＥＰＯ表达载体,转染ＣＯＳ－７细胞后获得高效表达。利用自行研制的小鼠抗人ＥＰＯ单抗及兔抗人ＥＰＯ多抗,对表达产物进行ＥＬＩＳＡ定量测定,细胞分泌ＥＰＯ量高达２５１±７Ｕ／ｍｌ．Ｋｒｙｓｔａｌ法测得体外生物活性２４１．５±６．５Ｕ／ｍｌ．用ＥＰＯ单抗免疫沉淀结合ＳＤＳ－ＰＡＧＥ对转染细胞的表达产物做了进一步鉴定,清晰地看到了ＥＰＯ条带。从高效表达ＥＰＯ的转染细胞中分离纯化ｍＲＮＡ,用ＲＴ－ＰＣＲ方法扩增并克隆到ＥＰＯ的ｃＤＮＡ,这为ＥＰＯ在其它系统中的表达及ＥＰＯ的功能与结构的研究打下了基础。     

Localization of Microtubule-Associated Protein (MAP) 1B in the Postsynaptic Densities of the Rat Cerebral Cortex

1. Although microtubule-associated protein (MAP) 1B and its phosphorylation have been suggested to be important for synapse formation among cortical neurons, the localization of MAP1B in synapses has not yet been confirmed. In this report, we examine the localization of MAP1B in synaptic regions. 2. The localization of MAP1B was observed by immunohistochemical and electron microscopic techniques using specific antibodies against MAP1B. 3. MAP1B immunoreactivities were widely distributed in the cerebral cortex and were observed in the postsynaptic area but not in presynaptic terminals. 4. These synapses were classified as the asymmetrical type. 5. Only some synapses exhibited MAP1B immunoreactivities. MAP1B-immunopositive synapses accounted for about half of the total synapses. 6. Such a localization suggests MAP1B's important roles in synaptic functions.     

人牛精浆蛋白相关新基因的cDNA克隆、定位和表达

为了研究牛精浆 (bovineseminalplasma ,BSP)蛋白及其相关蛋白在受精及受精卵发育中的重要作用 ,寻找BSP蛋白相关新基因 .采用cDNA末端快速扩增 (RACE)技术 ,克隆了一个BSP蛋白相关基因的cDNA序列 .应用辐射杂种细胞系 (RH)技术进行了基因染色体定位 .通过RT PCR检测了该基因在人体各组织中的表达情况 .并将该基因编码的蛋白进行了原核表达 .新基因的cDNA长度为 10 5 2bp ,其开放阅读框架 (ORF)编码了一个含 2 2 3个氨基酸残基的蛋白质 ,氨基酸序列中含有 4个纤连蛋白Ⅱ结构域 ,与BSP蛋白在结构上具有一定的相似性 ,称其为人BSP相关蛋白 (humanBSP relatedproteins ,HBRP) .该基因定位于染色体 19q13,在大肠杆菌中表达为 5 2kD的融合蛋白 .研究结果提示 ,应用RACE方法克隆了一种新的人类与BSP蛋白相关的基因 ,推测其编码蛋白是与BSP蛋白功能相关的结合蛋白 ,通过基因重组技术大量获得表达蛋白 ,对进一步研究新蛋白的生物学功能具有重要的意义 .