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At least 649 records · Page 36Linked to original sources

PubMed1998

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.

Cell Polarity↗

PubMed1992

Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones.

We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.

Bacteriophage lambda↗

PubMed2004

[Use PCR synthesis large fragment DNA].

Synthesis Large Fragment DNA using PCR (SLFD PCR) is a useful method to synthesize large fragment DNA. Use a known 500 approximately 600 basepair DNA fragment as PCR template, a series of 5' terminal primers are designed, and these primers are overlap one by one from 5' terminal to 3' terminal, the net DNA is just what you want to synthesize. The last 3' terminal primer of these primers has a BamH I site, and behind the BamH I site there are 15 bp overlap the 5' terminal of the template. Another downstream primer complement the 3' terminal of the template, and has a BamH I site too. PCR begin using the innerest 5' terminal primer and the downstream primer. After 10 cycles of PCR, use the product of the PCR as the template of next round PCR, but this time the upstream primer change to the 5' terminal outer primer . So do the PCRs, till all the 5' terminal primers take part in the PCR. Clone the final PCR product and BamH I cut the original DNA template, The DNA synthesis complete. It's a useful method to synthesize 100 approximately 200 bp DNA fragment, even more long DNA fragment.

Antimicrobial Cationic Peptides↗

PubMed1992

Localization of the human insulin-like growth-factor-binding protein 4 gene to chromosomal region 17q12-21.1.

Insulin-like growth-factor-binding proteins (IGFBPs) constitute a family of structurally related proteins that specifically bind insulin-like growth factors and modulate their functions. In this study, the chromosomal localization was determined for the gene encoding IGFBP4, i.e. inhibitory-IGFBP. A polymerase chain reaction (PCR) fragment corresponding to the previously published cDNA sequence was used to isolate overlapping cosmid clones. By fluorescent in situ hybridization to metaphase chromosomes, the IGFBP4 gene was then localized to chromosomal region 17q21-21.1. This result was in agreement with PCR analysis of a panel of somatic cell hybrids.

Base Sequence↗

PubMed2004

Molecular cloning of TSARG6 gene related to apoptosis in human spermatogenic cells.

Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

Amino Acid Sequence↗

PubMed1996

Generation of an integrated transcription map of the BRCA2 region on chromosome 13q12-q13.

An integrated approach involving physical mapping, identification of transcribed sequences, and computational analysis of genomic sequence was used to generate a detailed transcription map of the 1. 0-Mb region containing the breast cancer susceptibility locus BRCA2 on chromosome 13q12-q13. This region is included in the genetic interval bounded by D13S1444 and D13S310. Retrieved sequences from exon amplification or hybrid selection procedures were grouped into physical intervals and subsequently grouped into transcription units by clone overlap. Overlap was established by direct hybridization, cDNA library screening, PCR cDNA linking (island hopping), and/or sequence alignment. Extensive genomic sequencing was performed in an effort to understand transcription unit organization. In total, approximately 500 kb of genomic sequence was completed. The transcription units were further characterized by hybridization to RNA from a series of human tissues. Evidence for seven genes, two putative pseudogenes, and nine additional putative transcription units was obtained. One of the transcription units was recently identified as BRCA2 but all others are novel genes of unknown function as only limited alignment to sequences in public databases was observed. One large gene with a transcript size of 10.7 kb showed significant similarity to a gene predicted by the Caenorhabditis elegans genome and the Saccharomyces cerevisiae genome sequencing efforts, while another contained a motif sequence similar to the human 2',3' cyclic nucleotide 3' phosphodiesterase gene. Several retrieved transcribed sequences were not aligned into transcription units because no corresponding cDNAs were obtained when screening libraries or because of a lack of definitive evidence for splicing signals or putative coding sequence based on computational analysis. However, the presence of additional genes in the BRCA2 interval is suggested as groups of putative exons and hybrid selected clones that were transcribed in consistent orientations could be localized to common physical intervals.

BRCA2 Protein↗

PubMed2004

Characterization of a new PEPD allele causing prolidase deficiency in two unrelated patients: natural-occurrent mutations as a tool to investigate structure-function relationship.

Prolidase deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425-743) and PD4 (nt 661-973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the alpha2 domain of the "pita bread" structure proposed for E. coli and human prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.

Alleles↗

PubMed1991

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.

Bacteriophage lambda↗

PubMed1994

Cloning and characterization of the recA genes from Mycoplasma pulmonis and M. mycoides subsp. mycoides.

The RecA protein has a central role in DNA repair and is essential for homologous recombination in most eubacteria. Little is known about these critical processes in mycoplasmas. By using standard and inverse polymerase chain reactions (PCR) coupled with conventional cloning techniques, a series of overlapping fragments comprising the entire recA genes of Mycoplasma mycoides subsp. mycoides (Mm) and Mycoplasma pulmonis (Mp) were generated. Each gene was sequenced in its entirety. The recA genes of Mm and Mp would encode proteins of 345 amino acids (aa) and 339 aa, respectively. The mycoplasmal RecA proteins revealed strong conservation when compared with RecA sequences from other bacterial species.

Amino Acid Sequence↗

PubMed1995

Is the M1-muscarinic receptor a candidate gene for hypertension in the spontaneously hypertensive rat?

1. Central nervous system muscarinic receptors have been implicated in genetic models of hypertension as well as in stress adaptive cardiovascular responses. To determine if the sequence of the M1-muscarinic receptor is pathogenetic for hypertension, we analysed the differences between SHRLJ (spontaneously hypertensive rats) and WKYLJ (Wistar-Kyoto) rats in the sequence of the M1-receptor gene. 2. Specific primer sets were synthesized so as to cover the gene in 10 fragments (158-344 base pairs) with overlaps. Analysis was conducted by both polymerase chain reaction (PCR)-acrylamide and by single-stranded conformation polymorphism (SSCP). 3. No polymorphic locus was found by non-denaturing PCR-acrylamide nor by denaturing SSCP. Slight intrastrain variations in fragment 2 of the parental strains were examined by direct sequencing; however, no difference in sequence was found between SHRLJ and WKYLJ. To identify the chromosomal location and possible linkage with a polymorphic locus, rat/mouse somatic hybrid cell lines were examined. The rat M1-muscarinic receptor was assigned to chromosome 1. 4. Based on analysis of the coding sequence of the gene, our data do not support the hypothesis that the M1-muscarinic receptor is a candidate gene for hypertension. We conclude that, pending analyses of the promoter and regulatory regions of the gene, differences in the M1-muscarinic receptor in SHR either in receptor expression or in physiological response must be due to altered intracellular signalling or extracellular transynaptic events, but not due to a mutation of the gene sequence.

Animals↗

PubMed2002

Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors.

Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family, plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Here, we report that Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6, which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. We also show that the interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, we identified a specific DNA motif that is bound by Six3 and we demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. We also demonstrated that this interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae led to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos resulted in failure of lens placode invagination and production of delta-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development.

Amino Acid Motifs↗

PubMed2000

ABC transporter genes, kasKLM, responsible for self-resistance of a kasugamycin producer strain.

We previously reported that a 7.6-kb DNA fragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM acetyltransferase gene (kac338) and some other genes possibly involved in KSM biosynthesis. As an extension of that study, a 10-kb SacI-KpnI DNA fragment, located approximately 5-15-kb upstream of kac33, was cloned and a 4.2-kb SacI-EcoRI fragment therefrom was sequenced, revealing one incomplete (designated ORF J) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and M overlap one another with terminator/initiator ATGA sequence. RT-PCR analysis of a DNA region including kasKLM indicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATP-binding cassette (ABC) transporter superfamily. Homology search for the deduced KasK protein shows similarity to other ABC transporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasM genes encode different integral membrane proteins, both having six putative transmembrane helices. An expression plasmid for kasKLM (pTV-KLM) was constructed and these genes were expressed in E. coli JM 109, which had been sensitive to KSM. The transformant acquired resistance to KSM, suggesting that KasK, L and M proteins as a set in S. kasugaensis M338-M1 pump out KSM to protect the producer from its toxic metabolite.

ATP-Binding Cassette Transporters↗

PubMed2001

Identification, genomic structure, and screening of the vacuolar proton-ATPase membrane sector-associated protein M8-9 gene within the COD1 critical region (Xp11.4).

PURPOSE: Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4. METHODS: A complete physical contig of the COD1 region was partially sequenced and subjected to BLAST searches to identify homologies with GenBank ESTs. ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries. RACE was performed to complete the missing 5' end of the transcripts. Transcripts were compared with genomic sequences to specify intron-exon boundaries. Genomic DNAs from COD1-affected males from 3 families were screened for mutations using direct PCR sequencing of the exons. RESULTS: The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region. We confirmed its retinal expression and its genomic location in our physical contig. Eight exons (with flanking intronic sequences) were characterized from partial cDNA sequence and genomic sequence data. An additional 5' end exon was identified using RACE. No mutations were found in the COD1-affected males. CONCLUSIONS: The combination of disease mapping and information from the Human Genome project has enabled us to identify candidate genes within the COD1 region, including APT6M8-9 gene. We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome.

ATP-Binding Cassette Transporters↗

PubMed1991

Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease.

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5' end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

Base Sequence↗

PubMed1992

Cloning of the Huntington disease region in yeast artificial chromosomes.

The gene responsible for Huntington disease has been localized to a 2.5 million base pair (Mb) region between the loci D4S10 and D4S168 on the short arm of chromosome 4. As part of a strategy to clone the HD gene on the basis of its chromosomal location, we isolated genomic DNA from the HD region as a set of overlapping yeast artificial chromosome (YAC) clones. Twenty-eight YAC clones were identified by screening human YAC libraries with twelve PCR-based sequence-tagged sites (STSs) from the region. We assembled the YAC clones into overlapping sets by hybridizing them to a large number of DNA probes from the HD region, including the STSs. In addition, we isolated the ends of the human DNA inserts of most of the YAC clones to assist in the construction of the contig. Although almost half of the YACs appear to contain chimeric inserts and several contain internal deletions or other rearrangements, we were able to obtain over 2.2 Mb of the HD region in YACs, including one continuous segment of 2.0 Mb covering the region that most likely contains the HD gene. Ten of the twenty eight YAC clones comprise a minimal set spanning the 2.2 Mb. These clones provide reagents for the complete characterization of this region of the genome and for the eventual isolation of the HD gene.

Base Sequence↗

PubMed1993

Cloning and expression of cDNA encoding human galactocerebrosidase, the enzyme deficient in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide, a major lipid in myelin, kidney and epithelial cells of small intestine and colon. We describe the molecular cloning of human GALC cDNA and its expression in COS-1 cells. Degenerate PCR primers, derived from N-terminal amino acid sequence from the 51 kDa band from human brain, were used to amplify cat testes RNA, and the resulting product was used to screen human testes and brain libraries. Two overlapping clones contained the total protein coding region, while additional clones and PCR amplification were needed to obtain the complete 3' end of the cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007 bp of open reading frame (coding for 669 amino acids), and 1741 bp of 3' untranslated sequence. Modification of the sequence surrounding the initiation codon to one more favorable for expression, resulted in a 6-fold increase in GALC activity in transfected COS-1 cells. The isolation of this clone will permit investigations into the causes for GALC deficiency in humans and available animal models, development of more accurate tests for patient and carrier identification, and evaluation of methods for effectively treating GALC deficiency, initially using the animal models.

Amino Acid Sequence↗

PubMed1994

The first analysis of exon 1 (the transactivation domain) of the androgen receptor gene in infertile men with oligospermia or azoospermia.

OBJECTIVE: To examine the role of the androgen receptor (AR) gene in spermatogenesis by evaluating infertile men with idiopathic oligospermia or azoospermia, with special emphasis on the transactivation domain (exon 1) of AR gene because it has not been studied in this population previously. STUDY DESIGN: A molecular study of the AR gene. Deoxyribonucleic acid samples were screened for possible AR gene mutations using polymerase chain reactions (PCR). SETTING: The offices and laboratories of the Medical College of Georgia. PARTICIPANTS: Infertile men with oligospermia or azoospermia and an otherwise negative laboratory evaluation. Controls consisted of healthy fertile men. MAIN OUTCOME MEASURES: Each exon (2 to 8) and each of five overlapping exon segments for exon 1 of the AR gene was amplified using PCR for each participant's DNA sample. The PCR products were evaluated by size using electrophoresis and a DNA size marker. RESULTS: Sixteen idiopathic oligospermic or azoospermic men entered the study. All seven exons and the five overlapping segments of exon 1 were amplified and were of the appropriate size on electrophoresis when compared with controls, the DNA size marker, and the exon sequence. CONCLUSIONS: Preliminary protein studies on AR suggested that up to 40% of infertile men may have AR abnormalities. Since the availability of molecular analysis, no studies to date have evaluated the transcriptional activation domain (exon 1) of the AR gene in this population of infertile men. Our study found no gross AR mutations in the individuals studied. These results emphasize the importance of further studies needed to understand the regulation of spermatogenesis.

DNA↗

PubMed2006

Expression and sequences of genes encoding glutamate receptors and transporters in primate retina determined using 3'-end amplification polymerase chain reaction.

PURPOSE: Our long-term goal is to compare how expression of glutamate receptor and non-vesicular transporter subunits differs between single neurons in the primate retina. Here we set out to ascertain general expression in the retina of Macaca fascicularis using a robust technique suitable for both levels of analysis. We constructed full-complement cDNAs from whole retina RNA using a protocol optimized for detection of even low-abundance transcripts and for transcripts of various lengths. We probed these libraries for expression of genes encoding the AMPA- (GluR1-4) and kainate-sensitive (GluR5-7, KA1-2) ionotropic glutamate receptors, the metabotropic glutamate receptors (mGluR1-8), and five non-vesicular glutamate transporters (EAAT1-5) in the macaque retina and brain and determined large portions of coding sequences for each. We also asked whether each gene can be detected in cDNA generated from a limited amount of RNA extracted from an aldehyde-fixed retinal slice, a technique useful for probing gene expression in tissue used for histological studies. METHODS: We constructed full-length cDNA from RNA harvested from the macaque retina using a modified version of the 3'-end amplification (TPEA) technique of Dixon et al. With this technique, the 3' region is amplified arbitrarily using multiple primers to produce amplified cDNA containing as diverse and complete sample of genes as possible. We probed the cDNA for expression of glutamate receptors and non-vesicular transporters using gene-specific RT-PCR and assembled sequences from the reaction products using a series of overlapping primer pairs. We also used TPEA to compare expression in a small amount of RNA extracted from a fixed retinal slice. RESULTS: Macaque retinal cDNA created using TPEA contains a high abundance of transcripts of various lengths. Gene-specific PCR using primers designed against human sequences indicates expression for all GluR, mGluR, and non-vesicular transporter subunits for which we probed. Expression patterns were similar between two different macaque retinas, but different in brain. Several differences also exist between macaque and human brain. For some subunits, this is the first demonstration of expression in the macaque retina. The expression pattern obtained probing cDNA libraries generated from fixed tissue RNA with a different primer set was similar to that for fresh tissue. We sequenced between 1865 (mGluR8a) and 3697 (mGluR1) total nucleotides for each macaque gene and obtained complete coding sequences for GluR1-7, mGluR3 and mGluR4. A comparison with the corresponding human sequences reveal that the coding region of macaque GluR6 demonstrates the highest homology with only 26 nucleotide substitutions (99% homology), while mGluR4 demonstrates the lowest with 68 substitutions (97.5%). CONCLUSIONS: Neural tissue cDNA created using TPEA contains diverse transcripts of varying size, abundance, and homology. We established using TPEA the expression of all nine AMPA- and kainate-sensitive GluRs, all eight mGluRs and all five non-vesicular glutamate transporters and found that expression differs between macaque retina, macaque brain and human brain. The nucleotide sequences for the coding regions differed moderately between the human and macaque genes. We also found a similar expression pattern in a smaller amount of RNA extracted from a fixed retinal slice. Thus, this technique could be useful for comparing gene expression in cells extracted from fixed tissue pre-labeled using specific markers.

3' Flanking Region↗