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Copper is one of the most important trace elements to life. HumanHCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 ofE. coli. The whole opening reading frame ofHCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed inE. coli BL21 (DE3).

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.

After screening human fetal brain cDNA library, a novel human cDNA encoding an RNA-binding protein was cloned and namedGRY-RBP. HumanGRY-RBP cDNA is 2 932 bp in length, and encodes a protein of 623 amino acids with a deduced molecular mass of 69.6 ku. Human GRY-RBP contains three RNA recognition motifs (RRMs) and a glycine (G), tyrosin (Y)-rich C-terminus. Computer searches with sequence database revealed its moderate homology with RRMs of many RRM RNA-binding proteins. In this study, the full coding region and partial non-coding region of mouseGRY-RBP cDNA were amplified by PCR using mouse brain cDNA library as template. Three transcripts ofGry-rbp are expressed in all tissues as shown by Northern blot, but the amounts in heart, planceta and skeletal muscle are considerably higher than those in other tissues.

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A 1.2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5′ untranslated sequence and a 209 bp 3′ untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).

Genes encoding proteins with PHD (plant homeodomain) finger motif (C4HC3) are highly conserved fromArabidopis toHomo sapiens. One of the major functions of these genes is regulating the expression of homeotic genes during the stage of embryonic development. They play a role in cell-cycle and cell differentiation and seem to be related with some human malignant diseases, such as leukemia. A human placenta cDNA library has been screened with cDNA probe amplified by PCR. The PCR primers have been designed according to theM96 (a mouse gene encoding a protein with PHD domain) homologous data in dbEST. A 2.1 kb insert fragment in a positive cDNA clone has been isolated and sequenced. This new full-length cDNA is namedPHF2 (GenBank Acc: AF052205). The putative protein composed of 567 aa has two typical PHD fingers at its N-terminus. Meanwhile it is identified that there are several alternatively spliced transcripts ofPHF2 in different human tissues through the PCR amplification, Northern blot hybridization and analysis of genomic DNA structure.

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.

In order to learn the mechanism of brain development and differentiation, 90 expressed sequence tags (ESTs) were isolated by differential display from the cerebrum and cerebellum of 13-week and 33-week fetal brains. After searching database, 74 of them represented novel genes, some of them were homologous to the brain development related genes. Using total cDNA probes, 79 of the ESTs and their expression differences in fetal brain were further characterized.

Reticulons (RTN) are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are still elusive. In the present study, mouse reticulon 3 (mRTN3) is cloned and its expression pattern in a variety of tissues is investigated. Three alternatively spliced transcripts of 1.8, 2.8 and 4.2 kb are revealed by Northern blotting hybridization. The 1.8 and 2.8 kb transcripts are expressed in many tissues. The 2.8 kb transcript has a high level in brain and the 4.2 kb transcript is only found in brain. In situ hybridization and immunohisto-chemical analysis indicated its high expression in non-glial cells in some particular region of mouse central nervous system, such as hippocampus, sub-thalamus nucleus, thalamus nucleus and cerebrum cortex.