ABSTRACT
BACKGROUND: Urine testing for high-risk human papillomavirus (HR-HPV) detection could provide a non-invasive, simple method for cervical cancer screening. OBJECTIVES: We examined whether HR-HPV detection is affected by urine collection time, portion of urine stream, or urine fraction tested, and assessed the performance of HR-HPV testing in urine for detection of cervical intraepithelial neoplasia grade II or worse (CIN2+). STUDY DESIGN: A total of 37 female colposcopy clinic attendees, ≥ 30 years, provided three urine samples: "first void" urine collected at home, and "initial stream" and "mid-stream" urine samples collected at the clinic later in the day. Self- and physician-collected brush specimens were obtained at the same clinic visit. Colposcopy was performed and directed biopsies obtained if clinically indicated. For each urine sample, HR-HPV DNA testing was conducted for unfractionated, pellet, and supernatant fractions using the Trovagene test. HR-HPV mRNA testing was performed on brush specimens using the Aptima HPV assay. RESULTS: HR-HPV prevalence was similar in unfractionated and pellet fractions of all urine samples. For supernatant urine fractions, HR-HPV prevalence appeared lower in mid-stream urine (56.8%[40.8-72.7%]) than in initial stream urine (75.7%[61.9-89.5%]). Sensitivity of CIN2+ detection was identical for initial stream urine and physician-collected cervical specimen (89.9%[95%CI=62.7-99.6%]), and similar to self-collected vaginal specimen (79.1%[48.1-96.6%]). CONCLUSION: This is among the first studies to compare methodologies for collection and processing of urine for HR-HPV detection. HR-HPV prevalence was similar in first void and initial stream urine, and was highly sensitive for CIN2+ detection. Additional research in a larger and general screening population is needed.
Subject(s)
Early Detection of Cancer/methods , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Precancerous Conditions/diagnosis , Urine Specimen Collection/methods , Urine/virology , Uterine Cervical Dysplasia/diagnosis , Adult , DNA, Viral/isolation & purification , Female , Humans , Middle Aged , Papillomavirus Infections/complications , Pilot Projects , Precancerous Conditions/virology , RNA, Messenger/isolation & purification , RNA, Viral/isolation & purification , Sensitivity and Specificity , Uterine Cervical Dysplasia/virologyABSTRACT
BACKGROUND: The aim of neoadjuvant chemotherapy is to increase the likelihood of successful breast conservation surgery (BCS). Accurate identification of BCS candidates is a diagnostic challenge. Breast Cancer Index (BCI) predicts recurrence risk in estrogen receptor+lymph node-breast cancer. Performance of BCI to predict chemosensitivity based on pathological complete response (pCR) and BCS was assessed. METHODS: Real-time RT-PCR BCI assay was conducted using tumor samples from 150 breast cancer patients treated with neoadjuvant chemotherapy. Logistical regression and c-index were used to assess predictive strength and additive accuracy of BCI beyond clinicopathologic factors. RESULTS: BCI classified 42% of patients as low, 35% as intermediate and 23% as high risk. Low BCI risk group had 98.4% negative predictive value (NPV) for pCR and 86% NPV for BCS. High versus low BCI group had a 34 and 5.8 greater likelihood of achieving pCR and BCS, respectively (P=0.0055; P=0.0022). BCI increased c-index for pCR (0.875-0.924; P=0.017) and BCS prediction (0.788-0.843; P=0.027) beyond clinicopathologic factors. CONCLUSIONS: BCI significantly predicted pCR and BCS beyond clinicopathologic factors. High NPVs indicate that BCI could be a useful tool to identify breast cancer patients who are not eligible for neoadjuvant chemotherapy. These results suggest that BCI could be used to assess both chemosensitivity and eligibility for BCS.
Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/therapy , Adult , Aged , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Chemotherapy, Adjuvant , Female , Humans , Middle Aged , Neoadjuvant Therapy , Neoplasm Staging , Real-Time Polymerase Chain Reaction , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Retrospective StudiesABSTRACT
BACKGROUND: A dichotomous index combining two gene expression assays, HOXB13:IL17BR (H:I) and molecular grade index (MGI), was developed to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer. METHODS: In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the randomised prospective Stockholm trial, H:I and MGI were measured using real-time RT-PCR. Association with patient outcome was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling. RESULTS: The dichotomous H:I+MGI was significantly associated with distant recurrence and breast cancer death. The >50% of tamoxifen-treated patients categorised as low-risk had <3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorised as low risk with an 8.3% 10-year distant recurrence risk. CONCLUSION: Retrospective analysis of this randomised, prospective trial cohort validated the prognostic utility of H:I+MGI and was used to develop and test a continuous risk model that enables prediction of distant recurrence risk at the patient level.
Subject(s)
Breast Neoplasms/diagnosis , Homeodomain Proteins/analysis , Receptors, Interleukin/analysis , Biomarkers, Tumor/analysis , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Early Detection of Cancer , Female , Humans , Neoplasm Metastasis , Neoplasms, Hormone-Dependent/diagnosis , Postmenopause , Prognosis , Randomized Controlled Trials as Topic , Receptors, Interleukin-17 , Reverse Transcriptase Polymerase Chain Reaction , Risk Assessment , Sweden , Tamoxifen/therapeutic useABSTRACT
The recent cloning and characterization of the human histamine H(3) receptor cDNA marked a significant step toward a greater understanding of the role of this receptor in the central nervous system. We now report the cloning of the rat histamine H(3) receptor cDNA and demonstrate distinct pharmacological species differences. The rat cDNA clone encodes a putative 445-amino acid protein with 93% identity to the human H(3) receptor. Northern blot analysis revealed a major single entity of 2.7-kb in length expressed only in brain. Transfection of the rat receptor cDNA into SK-N-MC cells conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine and other H(3) agonists. N-[(3)H]methylhistamine saturably bound to transfected cells with high affinity (K(d) = 0.8 nM). All agonists tested had low or subnanomolar K(i) values similar to that for the human recombinant receptor. The antagonists thioperamide and clobenpropit also bound with high affinity (K(i) = 4 and 0.4 nM, respectively). This is in contrast to the antagonist profile obtained for the human recombinant receptor that showed K(i) values of 58 and 0.6 nM for thioperamide and clobenpropit, respectively. These data suggest that the low affinity of thioperamide for the human H(3) receptor represents a species difference in pharmacology and not a unique pharmacological subtype. It also was found that chloroproxyfan behaved as a full agonist at the rat recombinant receptor. These findings highlight the significance of validating the pharmacology of experimental compounds at both the rat and human H(3) receptors.
Subject(s)
Receptors, Histamine H3/drug effects , Adenylyl Cyclase Inhibitors , Amino Acid Sequence , Animals , Cloning, Molecular , Dose-Response Relationship, Drug , Humans , Methylhistamines/metabolism , Molecular Sequence Data , RNA, Messenger/analysis , Rats , Receptors, Histamine H3/chemistry , Receptors, Histamine H3/physiology , Recombinant Proteins/drug effectsABSTRACT
We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Profiling , NF-kappa B/genetics , Software , Transcription Factors/genetics , Automation , Base Sequence , Humans , Molecular Sequence Data , Nuclear Receptor Subfamily 4, Group A, Member 2 , RNA, Messenger/analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.
Subject(s)
Central Nervous System/chemistry , Hypothalamic Hormones/analysis , Neuropeptides/analysis , Receptors, Neuropeptide/analysis , Animals , Brain/metabolism , Brain Chemistry , CHO Cells , Cricetinae , Gene Expression , Hypothalamic Hormones/genetics , Hypothalamic Hormones/metabolism , In Situ Hybridization , Iodine Radioisotopes , Male , Neuropeptides/genetics , Neuropeptides/metabolism , Prolactin-Releasing Hormone , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/metabolism , Thalamic Nuclei/chemistry , Thyrotropin-Releasing Hormone/genetics , Tissue Distribution , Transfection , Tyrosine 3-Monooxygenase/analysisABSTRACT
Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.
Subject(s)
Conserved Sequence , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Genes, Plant , Multigene Family/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Base Sequence , Biomarkers/chemistry , CD3 Complex/physiology , Cell Differentiation/immunology , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Lymphocytes/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Organ Culture Techniques , Organ Specificity/genetics , Plant Proteins/genetics , RNA, Messenger/biosynthesis , Sequence Homology, Amino Acid , Signal Transduction/genetics , Signal Transduction/immunology , Thymus Gland/chemistry , Thymus Gland/metabolismABSTRACT
While homomers containing 5-HT(3A) subunits form functional ligand-gated serotonin (5-HT) receptors in heterologous expression systems (Jackson, M. B., and Yakel, J. L. (1995) Annu. Rev. Physiol. 57, 447-468; Lambert, J. J., Peters, J. A., and Hope, A. G. (1995) in Ligand-Voltage-Gated Ion Channels (North, R., ed) pp. 177-211, CRC Press, Inc., Boca Raton, FL), it has been proposed that native receptors may exist as heteromers (Fletcher, S., and Barnes, N. M. (1998) Trends Pharmacol. Sci. 19, 212-215). We report the cloning of a subunit 5-HT(3B) with approximately 44% amino acid identity to 5-HT(3A) that specifically modified 5-HT(3A) receptor kinetics, voltage dependence, and pharmacology. Co-expression of 5-HT(3B) with 5-HT(3A) modified the duration of 5-HT(3) receptor agonist-induced responses, linearized the current-voltage relationship, increased agonist and antagonist affinity, and reduced cooperativity between subunits. Reverse transcriptase-polymerase chain reaction in situ hybridization revealed co-localization of both 5-HT(3B) and 5-HT(3A) in a population of neurons in the amygdala, telencephalon, and entorhinal cortex. Furthermore, 5-HT(3A) and 5-HT(3B) mRNAs were expressed in spleen and intestine. Our data suggest that 5-HT(3B) might contribute to tissue-specific functional changes in 5-HT(3)-mediated signaling and/or modulation.
Subject(s)
Brain/metabolism , Gene Expression Regulation , Neurons/metabolism , Receptors, Serotonin/physiology , Serotonin Receptor Agonists/pharmacology , Serotonin/pharmacology , Amygdala/metabolism , Animals , Base Sequence , Biguanides/pharmacology , Calcium/metabolism , Cell Line , Cloning, Molecular , Entorhinal Cortex/metabolism , Female , Humans , Kinetics , Macromolecular Substances , Membrane Potentials/drug effects , Molecular Sequence Data , Oocytes/drug effects , Oocytes/physiology , Open Reading Frames , Receptors, Serotonin/chemistry , Receptors, Serotonin/genetics , Receptors, Serotonin, 5-HT3 , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Telencephalon/metabolism , Transcription, Genetic , Transfection , Xenopus laevisABSTRACT
We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.
Subject(s)
Cytomegalovirus/genetics , DNA, Viral/analysis , Genome, Viral , Oligonucleotide Array Sequence Analysis , Base Sequence , Cell Line , Gene Expression/drug effects , Humans , Kinetics , Molecular Sequence DataABSTRACT
Histamine regulates neurotransmitter release in the central and peripheral nervous systems through H3 presynaptic receptors. The existence of the histamine H3 receptor was demonstrated pharmacologically 15 years ago, yet despite intensive efforts, its molecular identity has remained elusive. As part of a directed effort to discover novel G protein-coupled receptors through homology searching of expressed sequence tag databases, we identified a partial clone (GPCR97) that had significant homology to biogenic amine receptors. The GPCR97 clone was used to probe a human thalamus library, which resulted in the isolation of a full-length clone encoding a putative G protein-coupled receptor. Homology analysis showed the highest similarity to M2 muscarinic acetylcholine receptors and overall low homology to all other biogenic amine receptors. Transfection of GPCR97 into a variety of cell lines conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine, but not to acetylcholine or any other biogenic amine. Subsequent analysis revealed a pharmacological profile practically indistinguishable from that for the histamine H3 receptor. In situ hybridization in rat brain revealed high levels of mRNA in all neuronal systems (such as the cerebral cortex, the thalamus, and the caudate nucleus) previously associated with H3 receptor function. Its widespread and abundant neuronal expression in the brain highlights the significance of histamine as a general neurotransmitter modulator. The availability of the human H3 receptor cDNA should greatly aid in the development of chemical and biological reagents, allowing a greater appreciation of the role of histamine in brain function.
Subject(s)
Receptors, Histamine H3/genetics , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Cells, Cultured , Central Nervous System/metabolism , Cloning, Molecular , DNA, Complementary/analysis , Histamine/metabolism , Humans , Methylhistamines/metabolism , Molecular Sequence Data , Rats , Receptors, Histamine H3/biosynthesis , Receptors, Neurotransmitter/biosynthesis , Receptors, Neurotransmitter/genetics , Sequence Homology, Amino Acid , Thymus Gland/physiology , TritiumSubject(s)
Gene Expression , Neurons/classification , Animals , Databases, Factual , Female , Gene Amplification , Humans , Lasers , Neurons/metabolism , RNA , Rats , Rats, Sprague-DawleyABSTRACT
We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.
Subject(s)
CD55 Antigens/genetics , Gene Expression Regulation, Developmental , Neural Crest/cytology , Neurons/cytology , CD55 Antigens/biosynthesis , CD55 Antigens/immunology , Carcinogens/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , Gene Expression Regulation, Developmental/drug effects , Humans , Neural Crest/immunology , Neurons/immunology , Tetradecanoylphorbol Acetate/pharmacology , Up-RegulationABSTRACT
The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.
Subject(s)
Cloning, Molecular/methods , RNA/genetics , DNA Primers/genetics , DNA, Complementary/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression/genetics , Nucleic Acid Hybridization/genetics , Phosphorus Radioisotopes , Viral ProteinsABSTRACT
The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.
Subject(s)
Cloning, Molecular/methods , DNA, Complementary/isolation & purification , Gene Amplification , RNA/genetics , DNA, Complementary/biosynthesis , False Positive Reactions , HeLa Cells , HumansABSTRACT
Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.
Subject(s)
Cloning, Molecular/methods , Gene Expression/genetics , RNA, Messenger/biosynthesis , Antiviral Agents/pharmacology , Blotting, Northern , Cell Count , DNA, Complementary/genetics , Gene Library , HeLa Cells , Humans , Interferon-gamma/pharmacology , Nucleic Acid Hybridization/methods , RNA, Messenger/analysis , Ribosomal Proteins/chemistry , Ribosomal Proteins/geneticsABSTRACT
We used subtractive hybridization to isolate clones of gamma 7, a 68 residue G-protein gamma subunit. Northern blotting and in situ hybridization reveal that the gamma 7 subunit mRNA is expressed primarily in medium-sized neurons of the neostriatum and nucleus accumbens and neurons of the olfactory tubercle, and at low levels in the dentate gyrus of the hippocampal formation and laminae II-III, and V of the neocortex. The gamma 7 mRNA is translocated into dendrites of neurons in the neostriatum and the dentate gyrus of the hippocampus. gamma 7 is expressed at relatively very low concentrations in peripheral tissues. The selective pattern of gamma 7 expression within the brain is highly reminiscent of those of the striatum-enriched adenylyl cyclase ACST, dopamine receptors, and the alpha subunit of G(olf), suggesting that, in striatum, gamma 7 may be a subunit of a G(olf) alpha-containing G protein that couples dopamine receptors selectively to ACST.
Subject(s)
GTP-Binding Proteins/biosynthesis , Gene Expression Regulation , Neostriatum/metabolism , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Size , DNA, Complementary/genetics , Dendrites/metabolism , Digoxigenin , GTP-Binding Proteins/genetics , Hippocampus/cytology , Hippocampus/metabolism , Male , Mice , Molecular Sequence Data , Neostriatum/cytology , Nerve Tissue Proteins/genetics , Nucleic Acid Hybridization , Nucleus Accumbens/cytology , Nucleus Accumbens/metabolism , Olfactory Pathways/cytology , Olfactory Pathways/metabolism , Organ Specificity , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, Dopamine/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
We report an improved subtractive cDNA cloning procedure, named "directional tag PCR subtraction," for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5' and 3' ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.
Subject(s)
Cloning, Molecular/methods , Corpus Striatum/chemistry , RNA, Messenger/isolation & purification , Animals , Base Sequence , Blotting, Southern , DNA/analysis , DNA Probes , Gene Expression , Gene Library , In Situ Hybridization , Molecular Sequence Data , Nerve Tissue Proteins/analysis , Polymerase Chain Reaction/methods , Rats , Rats, Sprague-DawleyABSTRACT
We have used RFLP analysis on DNA from a panel of interspecific (C57BL/6J x Mus spretus) F1 x M. spretus backcross offspring to assign the genes encoding 10 neuron-specific mRNAs and 2 loci corresponding to cyclophilin 2-related sequences to the mouse chromosomal map. The Pss1 locus encoding the forebrain-enriched protein kinase C substrate RC3, a component of dendritic spines, mapped to proximal Chr 9. The Camkl locus encoding the calmodulin-binding protein kinase-like vesicle protein 1G5 mapped to distal Chr 9. The Gng7 locus encoding the gamma 7 G-protein subunit, highly enriched in the striatum and presumptively coupled to dopamine receptors, mapped to mid-Chr 10. The Htr1f, Htr5a, Htr5b, and Htr7loci, encoding four serotonin receptors, mapped to Chr 16, 5, 1, and 19, respectively. The Peplb locus, encoding a CD26 ectopeptidase-like neuronal membrane protein activated by kainate and long-term potentiation, mapped to Chr 5. The D2Sut1e and Cpu3 loci, encoding neural proteins of unknown functions, mapped to Chrs 2 and 9, respectively. Two cyclophilin 2-related loci, Cphn2-r1 and Cphn2-r2, mapped to different regions of Chr 9. Comparison of these 12 newly mapped loci with the existing mouse map and known regions of syntenic homology with the human map, along with the known features and expression profiles of the products of these genes, suggests a few candidates for mouse mutations and human neurological and immunological deficits, including the Tourette syndrome and Bloom syndrome genes.
Subject(s)
Central Nervous System/metabolism , Chromosome Mapping , Mice/genetics , Nerve Tissue Proteins/genetics , Animals , Brain/metabolism , Crosses, Genetic , Female , Gene Expression Regulation , Hybridization, Genetic , Male , Mice/metabolism , Mice, Inbred C57BL , Muridae/genetics , Nerve Tissue Proteins/biosynthesis , Organ Specificity , Polymorphism, Restriction Fragment LengthABSTRACT
We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.