Ubiquitin-proteasomal regulation of chromatin remodeler INO80 in the nucleus accumbens mediates persistent cocaine craving.

Neuroadaptations in the nucleus accumbens (NAc) underlie cue-induced cocaine craving that intensifies ("incubates") during abstinence and is believed to contribute to persistent relapse vulnerability. Changes in gene expression often govern perpetual behavioral abnormalities, but epigenetic plasticity during prolonged abstinence from drug exposure is poorly understood. We examined how E3 ubiquitin ligase TRIM3 dysregulates chromatin remodeler INO80 to mediate cocaine craving during prolonged abstinence. We found that INO80 expression increased in the NAc on abstinence day 30 (AD30) but not on AD1 following cocaine self-administration. Furthermore, TRIM3, which mediates degradation of INO80, was reduced on AD30, along with TRIM3-INO80 interaction. Viral-mediated gene transfer of INO80 or TRIM3 governed cocaine craving during prolonged abstinence. Lastly, chromatin immunoprecipitation followed by massively parallel DNA sequencing identified INO80-mediated transcriptional regulation of predicted pathways associated with cocaine plasticity. Together, these results demonstrate a novel ubiquitin-proteasomal-epigenetic mechanism by which TRIM3-INO80 mediates cocaine craving during prolonged abstinence.

MEF2C transcription factor is associated with the genetic and epigenetic risk architecture of schizophrenia and improves cognition in mice.

Large-scale consortia mapping the genomic risk architectures of schizophrenia provide vast amounts of molecular information, with largely unexplored therapeutic potential. We harnessed publically available information from the Psychiatric Genomics Consortium, and report myocyte enhancer factor 2C (MEF2C) motif enrichment in sequences surrounding the top scoring single-nucleotide polymorphisms within risk loci contributing by individual small effect to disease heritability. Chromatin profiling at base-pair resolution in neuronal nucleosomes extracted from prefrontal cortex of 34 subjects, including 17 cases diagnosed with schizophrenia, revealed MEF2C motif enrichment within cis-regulatory sequences, including neuron-specific promoters and superenhancers, affected by histone H3K4 hypermethylation in disease cases. Vector-induced short- and long-term Mef2c upregulation in mouse prefrontal projection neurons consistently resulted in enhanced cognitive performance in working memory and object recognition paradigms at baseline and after psychotogenic drug challenge, in conjunction with remodeling of local connectivity. Neuronal genome tagging in vivo by Mef2c-Dam adenine methyltransferase fusion protein confirmed the link between cognitive enhancement and MEF2C occupancy at promoters harboring canonical and variant MEF2C motifs. The multilayered integrative approaches presented here provide a roadmap to uncover the therapeutic potential of transcriptional regulators for schizophrenia and related disorders.

The dendritic spine morphogenic effects of repeated cocaine use occur through the regulation of serum response factor signaling.

The nucleus accumbens (NAc) is a primary brain reward region composed predominantly of medium spiny neurons (MSNs). In response to early withdrawal from repeated cocaine administration, de novo dendritic spine formation occurs in NAc MSNs. Much evidence indicates that this new spine formation facilitates the rewarding properties of cocaine. Early withdrawal from repeated cocaine also produces dramatic alterations in the transcriptome of NAc MSNs, but how such alterations influence cocaine's effects on dendritic spine formation remain unclear. Studies in non-neuronal cells indicate that actin cytoskeletal regulatory pathways in nuclei have a direct role in the regulation of gene transcription in part by controlling the access of co-activators to their transcription factor partners. In particular, actin state dictates the interaction between the serum response factor (SRF) transcription factor and one of its principal co-activators, MAL. Here we show that cocaine induces alterations in nuclear F-actin signaling pathways in the NAc with associated changes in the nuclear subcellular localization of SRF and MAL. Using in vivo optogenetics, the brain region-specific inputs to the NAc that mediate these nuclear changes are investigated. Finally, we demonstrate that regulated SRF expression, in turn, is critical for the effects of cocaine on dendritic spine formation and for cocaine-mediated behavioral sensitization. Collectively, these findings reveal a mechanism by which nuclear-based changes influence the structure of NAc MSNs in response to cocaine.

Normalizing dopamine D2 receptor-mediated responses in D2 null mutant mice by virus-mediated receptor restoration: comparing D2L and D2S.

D2 receptor null mutant (Drd2(-/-)) mice have altered responses to the rewarding and locomotor effects of psychostimulant drugs, which is evidence of a necessary role for D2 receptors in these behaviors. Furthermore, work with mice that constitutively express only the D2 receptor short form (D2S), as a result of genetic deletion of the long form (D2L), provides the basis for a current model in which D2L is thought to be the postsynaptic D2 receptor on medium spiny neurons in the basal forebrain, and D2S the autoreceptor that regulates the activity of dopamine neurons and dopamine synthesis and release. Because constitutive genetic deletion of the D2 or D2L receptor may cause compensatory changes that influence functional outcomes, our approach is to identify aspects of the abnormal phenotype of a Drd2(-/-) mouse that can be normalized by virus-mediated D2 receptor expression. Drd2(-/-) mice are deficient in basal and methamphetamine-induced locomotor activation and lack D2 receptor agonist-induced activation of G protein-regulated inward rectifying potassium channels (GIRKs) in dopaminergic neurons. Here we show that virus-mediated expression of D2L in the nucleus accumbens significantly restored methamphetamine-induced locomotor activation, but not basal locomotor activity, compared to mice receiving the control virus. It also restored the effect of methamphetamine to decrease time spent in the center of the activity chamber in female but not male Drd2(-/-) mice. Furthermore, the effect of expression of D2S was indistinguishable from D2L. Similarly, virus-mediated expression of either D2S or D2L in substantia nigra neurons restored D2 agonist-induced activation of GIRKs. In this acute expression system, the alternatively spliced forms of the D2 receptor appear to be equally capable of acting as postsynaptic receptors and autoreceptors.

Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

Transient viral-mediated overexpression of alpha-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence.

Calcium/calmodulin-dependent protein kinase II (CaMKII) activity is necessary for the long-lasting expression of locomotor sensitization and enhanced drug-taking observed in rats previously exposed to psychostimulants. Exposure to these drugs also transiently increases alphaCaMKII levels in the nucleus accumbens (NAcc), an effect that, when mimicked by transient viral-mediated overexpression of alphaCaMKII in NAcc shell neurons, leads to long-lasting enhancement in locomotor responding to amphetamine and NAcc alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA). The present experiments characterized the dopamine (DA) dependence of the functional AMPA receptor upregulation observed long after transient overexpression of alphaCaMKII. Rats infected with herpes simplex virus-alphaCaMKII in the NAcc shell showed a transient increase in alphaCaMKII levels that peaked at 4 days post-infection and returned to baseline 8 days later. When challenged with AMPA (0.8 nmol/side) in the NAcc shell at 20 days post-infection, these rats showed enhanced locomotion compared with controls. This sensitized locomotor response was blocked when AMPA was coinfused with either the DA type-1 receptor antagonist SCH23390 (0.8 nmol/side) or the protein kinase A inhibitor Rp-cAMPS (80 nmol/side). Neither SCH23390 nor Rp-cAMPS produced locomotor effects when infused by itself into the NAcc shell. Furthermore, these antagonists did not block the acute non-sensitized locomotor response to AMPA observed in control rats. These findings show that transient viral-mediated overexpression of alphaCaMKII in neurons of the NAcc shell leads to long-lasting functional upregulation of AMPA receptors that is DA type-1 receptor and protein kinase A dependent. Thus, transient increases in levels of alphaCaMKII in the NAcc shell produce long-lasting changes in the way that DA and glutamate interact in this site to generate locomotor behavior.

beta-Secretase cleavage of the amyloid precursor protein mediates neuronal apoptosis caused by familial Alzheimer's disease mutations.

The amyloid precursor protein (APP) is cleaved by two enzymes, beta-secretase and gamma-secretase, to generate the pathological amyloid beta (Abeta) peptide. Expression of familial Alzheimer's disease (FAD) mutants of APP in primary neurons causes both intracellular accumulation of the C-terminal beta-secretase cleavage product of APP and increased secretion of Abeta, and eventually results in apoptotic death of the cells. To determine whether either of these two processing products of APP is involved in this apoptotic pathway, we first modeled experimentally the accumulation of the beta-secretase cleavage product in neurons. The C-terminal 100 amino acids (C100) of APP, with and without a signal peptide, was expressed in cells via recombinant herpes simplex virus (HSV) vectors. Both transgene products were targeted to the membrane, and both caused apoptosis in the neurons, implicating the beta-secretase cleavage product of APP in apoptosis caused by FAD APPs. Expression in neurons of a mutant of FAD APP that inhibited beta-secretase cleavage inhibited its ability to cause apoptosis. However, expression in neurons of a mutant of FAD APP that inhibited gamma-secretase cleavage did not inhibit the ability of this mutant to cause apoptosis. These data suggested that the C-terminal beta-secretase cleavage product of APP, but not Abeta, mediates the apoptosis caused by FAD mutants of APP. Consistent with this hypothesis, C31, which is generated from the beta-secretase cleavage product, itself caused neuronal apoptosis. Inhibitors of caspases 3, 6 and 8, but not of caspase 9, inhibited the apoptosis caused by FAD mutants of APP. It may be inferred from these data that beta-secretase cleavage of FAD mutants of APP allows the appropriate caspase access to its site of action to produce C31, which directly causes neuronal apoptosis.

Dopamine D2 receptor-induced heterologous sensitization of adenylyl cyclase requires Galphas: characterization of Galphas-insensitive mutants of adenylyl cyclase V.

Whereas acute stimulation of Galphai/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Galphas-coupled receptors. Galphas-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Galphas-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D2L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D2 receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Galphas-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D2L receptor demonstrated that all three mutants had little or no responsiveness to beta-adrenergic receptor-mediated activation of Galphas but that the mutants retained sensitivity to forskolin and to D2L receptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Galphas-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Galphas is required for the expression of heterologous sensitization.

Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated cAMP response element-binding protein expression in nucleus accumbens.

Drugs of abuse regulate the transcription factor cAMP response element-binding protein (CREB) in striatal regions, including the nucleus accumbens (NAc). To explore how regulation of CREB in the NAc affects behavior, we used herpes simplex virus (HSV) vectors to elevate CREB expression in this region or to overexpress a dominant-negative mutant CREB (mCREB) that blocks CREB function. Rats treated with HSV-mCREB in place conditioning studies spent more time in environments associated with cocaine, indicating increased cocaine reward. Conversely, rats treated with HSV-CREB spent less time in cocaine-associated environments, indicating increased cocaine aversion. Studies in which drug-environment pairings were varied to coincide with either the early or late effects of cocaine suggest that CREB-associated place aversions reflect increased cocaine withdrawal. Because cocaine withdrawal can be accompanied by symptoms of depression, we examined how altered CREB function in the NAc affects behavior in the forced swim test (FST). Elevated CREB expression increased immobility in the FST, an effect that is opposite to that caused by standard antidepressants and is consistent with a link between CREB and dysphoria. Conversely, overexpression of mCREB decreased immobility, an effect similar to that caused by antidepressants. Moreover, the kappa opioid receptor antagonist nor-Binaltorphimine decreased immobility in HSV-CREB- and HSV-mCREB-treated rats, suggesting that CREB-mediated induction of dynorphin (an endogenous kappa receptor ligand) contributes to immobility behavior in the FST. Exposure to the FST itself dramatically increased CREB function in the NAc. These findings raise the possibility that CREB-mediated transcription within the NAc regulates dysphoric states.

Alzheimer's disease: dysfunction of a signalling pathway mediated by the amyloid precursor protein?

All individuals with Alzheimer's disease (AD) experience a progressive loss of cognitive function, resulting from a neurodegenerative process characterized by the deposition of beta-amyloid (A beta) in plaques and in the cerebrovasculature, and by the formation of neurofibrillary tangles in neurons. The cause of the neuronal death is unknown but it is thought to be linked in some way to the beta-amyloid precursor protein (APP), which is the source of the A beta that accumulates in the AD brain. There are two pieces of supporting data for this: first, APP is overexpressed in Down's syndrome, which leads to AD-like neuropathology by the age of 40 in virtually all affected individuals; secondly, specific point mutations in APP cause some forms of familial AD. Our laboratory has focused on a specific aspect of APP and its connection with the neuronal destruction seen in AD. We have hypothesized that AD results from a progressive dysfunction of APP. In addition, on the basis of recent data generated by our laboratory and others, we propose that in the normal brain a percentage of APP is present as an integral protein of the plasma membrane that mediates the transduction of extracellular signals into the cell via its A beta-containing C-terminal tail. In AD, accumulation of abnormal levels of the C-terminus in the neuron disturbs this signal-transduction function of APP, causing disorders in the cell-cycle machinery and consequent apoptosis. Here, we discuss the key findings that support this hypothesis, and discuss its therapeutic implications for AD.

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin.

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

Expression of the cAMP response element binding protein (CREB) in hippocampus produces an antidepressant effect.

BACKGROUND: Recent studies have demonstrated that chronic antidepressant treatment increases the expression of the cyclic amp (cAMP) response element binding protein (CREB) in rat hippocampus. The study presented here was conducted to determine if CREB is a relevant target that produces an antidepressant-like effect. METHODS: We employed the herpes simplex virus (HSV)-mediated gene transfer technique to overexpress CREB and determined its effect on the learned helplessness and forced swim tests, two established models used for pharmacological screening of antidepressant drugs. RESULTS: In the learned helplessness model, rats that received bilateral microinjection of HSV-CREB into the dentate gyrus showed significantly fewer escape failures in the subsequent conditioned avoidance test than those injected with control vector (HSV-LacZ). In contrast, microinjection of HSV-CREB in either the CA1 pyramidal cell layer of hippocampus or the prefrontal cortex did not produce an antidepressant response. In the forced swim test, CREB expression in the dentate gyrus resulted in a significantly shorter immobility time than those injected with HSV-LacZ. CONCLUSIONS: These results demonstrate that over-expression of CREB in hippocampus results in an antidepressant effect and suggest that CREB may serve as a potential molecular target for novel therapeutic agents.

Overexpression of HuD accelerates neurite outgrowth and increases GAP-43 mRNA expression in cortical neurons and retinoic acid-induced embryonic stem cells in vitro.

The neuron-specific RNA-binding protein HuD binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and stabilizes the mRNA. We have previously shown that overexpression of HuD in PC12 cells increases GAP-43 protein expression and induces the spontaneous formation of multiple neurites (K. D. Anderson et al. 2000. J. Neurochem. 75: 1103-1114). In this study, we examined the effects of HuD overexpression on the initial stages of neurite outgrowth and on GAP-43 gene expression using two in vitro systems: E19 rat cortical neurons and retinoic acid (RA)-induced embryonic stem (ES) cells. Normal neurite outgrowth of cortical neurons in vitro occurs over a 3-day period with a concomitant increase in GAP-43 and HuD expression. Cortical cells were infected with a replication-deficient HSV-1 vector containing the HuD cDNA in the sense orientation (HSV-HuD). Overexpression of HuD accelerated the formation of neurites. Immunocytochemical analysis showed that excess HuD resulted in a threefold increase in the number of GAP-43-positive cells undergoing morphological differentiation after 24 h of treatment. Using in situ hybridization, we found that the increased HuD expression resulted in a twofold increase in the levels of GAP-43 mRNA. Similarly, overexpression of HuD in RA-induced embryonic stem cells was found to increase the number of GAP-43-positive cells undergoing process outgrowth. In conclusion, our results demonstrate that HuD functions in the initiation of neurite outgrowth in a manner due, at least in part, to its regulation of GAP-43 expression.

Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala.

At least two temporally and mechanistically distinct forms of memory are conserved across many species: short-term memory that persists minutes to hours after training and long-term memory (LTM) that persists days or longer. In general, repeated training trials presented with intervening rest intervals (spaced training) is more effective than massed training (the same number of training trials presented with no or short intervening rest intervals) in producing LTM. LTM requires de novo protein synthesis, and cAMP response element-binding protein (CREB) may be one of the transcription factors regulating the synthesis of new proteins necessary for the formation of LTM. Here we show that rats given massed fear conditioning training show no or weak LTM, as measured by fear-potentiated startle, compared with rats given the same amount of training but presented in a spaced manner. Increasing CREB levels specifically in the basolateral amygdala via viral vector-mediated gene transfer significantly increases LTM after massed fear training. The enhancing effect of CREB overexpression on LTM formation is shown to be specific in terms of biochemistry, anatomy, time course, and the training procedure used. These results suggest that CREB activity in the amygdala serves as a molecular switch for the formation of LTM in fear conditioning.

Overview of gene delivery into cells using HSV-1-based vectors.

This overview describes the considerations involved in the preparation and use of herpes simplex virus type 1 (HSV-1) as a vector for gene transfer into neurons. Strategies for gene delivery into neurons, either to study the molecular biology of brain function or for gene therapy, must utilize vectors that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system in vivo. This unit describes the biology of HSV-1 along with a discussion covering development of amplicon and genomic HSV-1 vectors. Advantages and disadvantages of current HSV-1 vectors are presented, and HSV-1 vectors are compared with other vectors for gene transfer into neurons.

Generation of high-titer defective HSV-1 vectors.

There are two types of replication-deficient herpes simplex virus type 1 (HSV-1) vectors: those in which the foreign DNA of interest is cloned into the viral genome itself, and those that are comprised of a plasmid (amplicon) carrying minimal HSV-1 sequences that allow it to be packaged into virus particles with the aid of a helper virus. This unit describes the generation of helper virus stocks. Preparation of recombinant amplicon vector particles by transfection of amplicon and superinfection of helper virus into cells, and harvesting of packaged particles, is also delineated. Thorough characterization of the viral vector stock involves measuring (1) the helper virus plaque-forming units per ml (pfu/ml) on 2-2 cells, (2) the wild-type HSV-1 pfu/ml on Vero cells, and (3) the amplicon stock infectious vector units per ml (ivu/ml) on PC12 cells. Support protocols detail methods for determining titers of helper virus and wild-type virus by plaque assay, and of amplicon stocks by vector assay.

Overview of neural gene expression.

This overview describes issues that must be considered before attempting to express neural cDNAs in mammalian cells, including the choice of expression vector and cell type. Considerations for introducing recombinant vectors into cells are discussed along with a comparison of achieving stable or transient expression. Finally, the appropriate promoter is crucial and must be chosen to fit the design of the expression system.

Conversion of mRNA into double-stranded cDNA.

Enzymatic conversion of mRNA into double-stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide-primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt-ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The Alternate Protocol is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The Alternate Protocol takes advantage of a linker-primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt-ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5-methyl-dCTP in the first-strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI/XhoI-digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker-primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.