Combat-related Mild Traumatic Brain Injury: Association between Baseline Diffusion-Tensor Imaging Findings and Long-term Outcomes.

Purpose To determine whether functional outcomes of veterans who sustained combat-related mild traumatic brain injury (TBI) are associated with scalar metrics derived from diffusion-tensor (DT) imaging at their initial postdeployment evaluation. Materials and Methods This HIPAA-compliant retrospective study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From 2010 to 2013, initial postdeployment evaluation, including clinical assessment and brain magnetic resonance (MR) examination with DT imaging, was performed in combat veterans who sustained mild TBI while deployed. Outcomes from chart review encompassed initial postdeployment clinical assessment as well as later functional status, including evaluation of occupational status and health care utilization. Scalar diffusion metrics from the initial postdeployment evaluation were compared with outcomes by using multivariate analysis. Veterans who did and did not return to work were also compared for differences in clinical variables by using t and χ(2) tests. Results Postdeployment evaluation was performed a mean of 3.8 years after injury (range, 0.5-9 years; standard deviation, 2.5 years). After a mean follow-up of 1.4 years (range, 0.5-2.5 years; standard deviation, 0.8 year), 34 of 57 veterans (60%) had returned to work. Return to work was associated with diffusion metrics in multiple regions of white matter, particularly in the left internal capsule and the left frontal lobe (P = .02-.05). Overall, veterans had a mean of 46 health care visits per year during the follow-up period (range, 3-196 visits per year; standard deviation, 41 visits per year). Cumulative health care visits over time were inversely correlated with diffusion anisotropy of the splenium of the corpus callosum and adjacent parietal white matter (P < .05). Clinical measures obtained during initial postdeployment evaluation were not predictive of later functional status (P = .12-.8). Conclusion Differences in white matter microstructure may partially account for the variance in functional outcomes among veterans who sustained combat-related mild TBI. (©) RSNA, 2016.

Individuals With Traumatic Brain Injury and Their Significant Others' Perceptions of Information Given About the Nature and Possible Consequences of Brain Injury: Analysis of a National Survey.

PURPOSE OF THE STUDY: An online survey was developed to assess how well individuals with brain injuries and family/friends of those with traumatic brain injury (TBI) (significant others) felt they were informed about the nature and consequences of brain injury. PARTICIPANTS: A total of 117 significant others completed the survey. They were primarily female (84.6%), white (94.9%), and well educated (81.2%). A total of 149 individuals with brain injuries completed the survey and again were primarily female (63.8%), white (88.2%), and well educated (82.9%). RESULTS: More than half of the significant other respondents indicated that they were not provided enough information about TBI (53.5%). Up to 53.8% of the respondents with TBI felt that they were not provided enough information, with 43% reporting dissatisfaction with services. Female survivors and those with mild brain injuries were significantly more likely to feel that they were not provided sufficient rehabilitation or information. Increased satisfaction with services was correlated with decreased time since injury (r = -0.165, p = .049). Qualitative analysis revealed key themes about prognostic information and the adequacy of discharge planning and resources. IMPLICATIONS FOR CASE MANAGEMENT PRACTICE: Given that more than half of all surveyed indicated that they were not well-informed about brain injury and its possible effects, it is evident that case managers and their teams need to be aware of and invested in their efforts to educate both individuals with brain injuries and their significant others (family and friends) about both the nature and consequences of brain injury. Specific recommendations for practice are included.

The adult respiratory distress syndrome cognitive outcomes study: long-term neuropsychological function in survivors of acute lung injury.

RATIONALE: Cognitive and psychiatric morbidity is common and potentially modifiable after acute lung injury (ALI). However, practical measures of neuropsychological function for use in multicenter trials are lacking. OBJECTIVES: To determine whether a validated telephone-based neuropsychological test battery is feasible in a multicenter trial. To determine the frequency and risk factors for long-term neuropsychological impairment. METHODS: As an adjunct study to the Acute Respiratory Distress Syndrome Clinical Trials Network Fluid and Catheter Treatment Trial, we assessed neuropsychological function at 2 and 12 months post-hospital discharge. MEASUREMENTS AND MAIN RESULTS: Of 406 eligible survivors, we approached 261 to participate and 213 consented. We tested 122 subjects at least once, including 102 subjects at 12 months. Memory, verbal fluency, and executive function were impaired in 13% (12 of 92), 16% (15 of 96), and 49% (37 of 76) of long-term survivors. Long-term cognitive impairment was present in 41 of the 75 (55%) survivors who completed cognitive testing. Depression, post-traumatic stress disorder, or anxiety was present in 36% (37 of 102), 39% (40 of 102), and 62% (63 of 102) of long-term survivors. Enrollment in a conservative fluid-management strategy (P = 0.005) was associated with cognitive impairment and lower partial pressure of arterial oxygen during the trial was associated with cognitive (P = 0.02) and psychiatric impairment (P = 0.02). CONCLUSIONS: Neuropsychological function can be assessed by telephone in a multicenter trial. Long-term neuropsychological impairment is common in survivors of ALI. Hypoxemia is a risk factor for long-term neuropsychological impairment. Fluid management strategy is a potential risk factor for long-term cognitive impairment; however, given the select population studied and an unclear mechanism, this finding requires confirmation.

Assessment scales for disorders of consciousness: evidence-based recommendations for clinical practice and research.

OBJECTIVES: To conduct a systematic review of behavioral assessment scales for disorders of consciousness (DOC); provide evidence-based recommendations for clinical use based on their content validity, reliability, diagnostic validity, and ability to predict functional outcomes; and provide research recommendations on DOC scale development and validation. DATA SOURCES: Articles published through March 31, 2009, using MEDLINE, CINAHL, Psychology and Behavioral Sciences Collection, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, Cochrane Central Register of Controlled Trials, Biomedical Reference Collection, and PsycINFO. Thirteen primary terms that defined DOC were paired with 30 secondary terms that defined aspects of measurement. Scale names, abbreviations, and authors were also used as search terms. Task force members identified additional articles by using personal knowledge and examination of references in reviewed articles. STUDY SELECTION: Primary criteria included the following: (1) provided reliability, diagnostic validity, and/or prognostic validity data; (2) examined a cohort, case control, or case series sample of persons with DOC who were age older than or equal to 18 years; and (3) assessed in an acute care or rehabilitation setting. Articles were excluded if peer review was not conducted, original data were not reported, or an English language article was not available. The initial search yielded 580 articles. After paired rater review of study abstracts, guideline development was based on 37 articles representing 13 DOC scales. DATA EXTRACTION: Rater pairs classified studies addressing diagnostic and prognostic validity by using the American Academy of Neurology 4-tier level of evidence scheme, and reliability by using a task force-developed 3-tier evidence scheme. An independent quality review of ratings was conducted, and corrections were made. DATA SYNTHESIS: The Coma Recovery Scale-Revised (CRS-R), Sensory Stimulation Assessment Measure (SSAM), Wessex Head Injury Matrix (WHIM), Western Neuro Sensory Stimulation Profile (WNSSP), Sensory Modality Assessment Technique (SMART), Disorders of Consciousness Scale (DOCS), and Coma/Near-Coma Scale (CNC) have acceptable standardized administration and scoring procedures. The CRS-R has excellent content validity and is the only scale to address all Aspen Workgroup criteria. The SMART, SSAM, WHIM, and WNSSP demonstrate good content validity, containing items that could distinguish persons who are in a vegetative state, are in a minimally conscious state (MCS), or have emerged from MCS. The Full Outline of UnResponsiveness Score (FOUR), WNSSP, CRS-R, Comprehensive Levels of Consciousness Scale (CLOCS), and Innsbruck Coma Scale (INNS) showed substantial evidence of internal consistency. The FOUR and the CRS-R showed substantial evidence of good interrater reliability. Evidence of diagnostic validity and prognostic validity in brain injury survivor samples had very high levels of potential bias because of methodologic issues such as lack of rater masking. CONCLUSIONS: The CRS-R may be used to assess DOC with minor reservations, and the SMART, WNSSP, SSAM, WHIM, and DOCS may be used to assess DOC with moderate reservations. The CNC may be used to assess DOC with major reservations. The FOUR, INNS, Glasgow-Liege Coma Scale, Swedish Reaction Level Scale-1985, Loewenstein Communication Scale, and CLOCS are not recommended at this time for bedside behavioral assessment of DOC because of a lack of content validity, lack of standardization, and/or unproven reliability.

Cognitive, mood and quality of life impairments in a select population of ARDS survivors.

BACKGROUND AND OBJECTIVE: There is increasing evidence that survivors of ARDS may have impairments in cognitive function, mood and quality of life. This study investigated associations between cognitive impairment, mood disorders and quality of life in a select group of ARDS survivors. METHODS: A cross-sectional study was conducted to describe the specific impairments in cognitive function, mood and quality of life in a group of 79 self-selected ARDS survivors who contacted an Internet-based support site. A battery of cognitive tests was administered by telephone interview. Standardized scores on cognitive tests were compared with normative values and tested for associations with indices of anxiety, depression and quality of life. RESULTS: Cognitive impairment was found in 56% of subjects. Compared with population norms, 24% of subjects had deficiencies in short-term memory (P = 0.04) and 29% in executive functioning (P = 0.001). Moderate or severe anxiety was present in 48% of the study population, and 34% had moderate or severe depression. Moderate or severe anxiety was present in 61% of subjects with evidence of cognitive impairment as compared with 31% of subjects without. Subjects with cognitive impairment scored worse than subjects without cognitive impairment on most subscales of the SF-36 and the Sickness Impact Profile questionnaire. CONCLUSIONS: Significant cognitive abnormalities may be present in long-term ARDS survivors, particularly in memory and executive function. Impairments in cognition appear to be associated with significantly increased anxiety and worse quality of life.

Formation and validation of a telephone battery to assess cognitive function in acute respiratory distress syndrome survivors.

PURPOSE: Describe initial development and validation of a test battery composed of established instruments designed to detect, via telephone interview, cognitive abnormalities in survivors of acute respiratory distress syndrome. METHODS: Two cross-sectional studies were performed, including the following phases: (1) initial battery construction, (2) feasibility, (3) item reduction, (4) convergent and divergent validity, and (5) telephone administration compared with in-face interviews in a separate population. RESULTS: There was a broad range of cognitive function detected in the derivation population, and all subjects completed the interview. There was convergence of cognitive impairment with moderate/severe anxiety (P = .008), the Sickness Impact Profile Psychosocial Summary Score (mean difference, 15.3; 95% CI, 7.74-22.9; P = .0001), and the mental health domains of the Short Form 36. Subjects with cognitive impairment had no detectable difference in the physical function domains of the Short Form 36. When administered to the validation population, telephone tests of memory, attention, reasoning, and executive functions had good intraclass correlation with the in-face interviews (P < .01). CONCLUSIONS: Detection of cognitive abnormalities in acute respiratory distress syndrome survivors using a telephone-administered test battery derived from standard cognitive tests is feasible and has evidence of construct validity. This battery may be useful as a research tool when in-face interviews are not feasible.

Cholinergic augmentation with donepezil enhances recovery in short-term memory and sustained attention after traumatic brain injury.

OBJECTIVE: To examine effects of donepezil on short-term memory and sustained attention in postacute patients with traumatic brain injury (TBI). DESIGN: A 24-week, randomized, placebo-controlled, double-blind crossover trial. SETTING: Outpatient clinics in 2 teaching hospitals. PARTICIPANTS: Eighteen postacute TBI patients with cognitive impairment. INTERVENTION: Patients were randomly assigned to group A or group B. Patients in group A received donepezil for the first 10 weeks and then a placebo for another 10 weeks. The 2 treatment phases were separated by a washout period of 4 weeks. Patients in group B received the preparations in the opposite order. MAIN OUTCOME MEASURES: Short-term memory and sustained attention were assessed by 2 indexes (Auditory Immediate Index [AII], Visual Immediate Index [VII]) of the Wechsler Memory Scale-III and the Paced Auditory Serial Addition Test (PASAT), at baseline, week 10, and week 24 of the trial. RESULTS: Intragroup comparison of different phases of the trial in both groups showed that donepezil significantly increased the testing scores of the AII and VII, as well as PASAT scores, compared with baseline. There was no significant change in the testing scores between assessment at baseline and the end of the placebo phase in group B. Intergroup comparison at the 10-week assessment showed significantly improved testing scores in group A with donepezil over group B with the placebo. The improved testing scores with donepezil in group A were sustained after the washout period and placebo phase, suggesting a carry-over effect of the medication. CONCLUSIONS: Donepezil increased neuropsychologic testing scores in short-term memory and sustained attention in postacute TBI patients. Cholinergic augmentation may be a viable approach to restore memory and attention after TBI.

Dynamic imaging in mild traumatic brain injury: support for the theory of medial temporal vulnerability.

OBJECTIVE: To determine whether patients with mild traumatic brain injury (TBI) and persistent postconcussive symptoms have evidence of temporal lobe injury on dynamic imaging. DESIGN: Case series. SETTING: An academic medical center. PARTICIPANTS: Twenty patients with a clinical diagnosis of mild TBI and persistent postconcussive symptoms were referred for neuropsychologic evaluation and dynamic imaging. Fifteen (75%) had normal magnetic resonance imaging (MRI) and/or computed tomography (CT) scans at the time of injury. INTERVENTIONS: Neuropsychologic testing, positron-emission tomography (PET), and single-photon emission-computed tomography (SPECT). MAIN OUTCOME MEASURES: Temporal lobe findings on static imaging (MRI, CT) and dynamic imaging (PET, SPECT); neuropsychologic test findings on measures of verbal and visual memory. RESULTS: Testing documented neurobehavioral deficits in 19 patients (95%). Dynamic imaging documented abnormal findings in 18 patients (90%). Fifteen patients (75%) had temporal lobe abnormalities on PET and SPECT (primarily in medial temporal regions); abnormal findings were bilateral in 10 patients (50%) and unilateral in 5 (25%). Six patients (30%) had frontal abnormalities, and 8 (40%) had nonfrontotemporal abnormalities. Correlations between neuropsychologic testing and dynamic imaging could be established but not consistently across the whole group. CONCLUSIONS: Patients with mild TBI and persistent postconcussive symptoms have a high incidence of temporal lobe injury (presumably involving the hippocampus and related structures), which may explain the frequent finding of memory disorders in this population. The abnormal temporal lobe findings on PET and SPECT in humans may be analogous to the neuropathologic evidence of medial temporal injury provided by animal studies after mild TBI.

The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model.

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.

Exposure of mammalian cells to ultraviolet (UV) light or high osmolarity strongly activates the c-Jun amino-terminal protein kinase (JNK) cascade, causing induction of many target genes. Exposure to UV light or osmotic shock induced clustering and internalization of cell surface receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF), and interleukin-1 (IL-1). Activation of the EGF and TNF receptors was also detected biochemically. Whereas activation of each receptor alone resulted in modest activation of JNK, coadministration of EGF, IL-1, and TNF resulted in a strong synergistic response equal to that caused by exposure to osmotic shock or UV light. Inhibition of clustering or receptor down-regulation attenuated both the osmotic shock and UV responses. Physical stresses may perturb the cell surface or alter receptor conformation, thereby subverting signaling pathways normally used by growth factors and cytokines.

Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.

Extracellular stimuli that activate the transcription factor NF-kappaB cause rapid phosphorylation of the IkappaBalpha inhibitor, which retains NF-kappaB in the cytoplasm of nonstimulated cells. Phosphorylation of IkappaBalpha is followed by its rapid degradation, the inhibition of which prevents NF-kappaB activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IkappaBalpha. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IkappaBalpha degradation. Substitutions of the homologous sites in IkappaBbeta, serines 19 and 23, also prevent inducible IkappaBbeta degradation. We suggest that activation of a single IkappaB kinas e or closely related IkappaB kinases is the first cr itical step in NF-kappaB activation. Once phosphorylated, IkappaB is ubiquitinated. Unlike wild-type IkappaBalpha, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IkappaBalpha without affecting its phosphorylation, polyubiquitination is required for inducible IkappaB degradation.

Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis.

Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but the mechanism underlying this activity has been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids.

Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B.

Cell shape changes exert specific effects on gene expression. It has been speculated that the cytoskeleton is responsible for converting changes in the cytoarchitecture to effects on gene transcription. However, the signal transduction pathways responsible for cytoskeletal-nuclear communication remained unknown. We now provide evidence that a variety of agents and conditions that depolymerize microtubules activate the sequence-specific transcription factor NF-kappa B and induce NF kappa B-dependent gene expression. These effects are caused by depolymerization of microtubule because they are blocked by the microtubule-stabilizing agent taxol. In nonstimulated cells, the majority of NF-kappa B resides in the cytosplasm as a complex with its inhibitor I kappa B. Upon cell stimulation, NF-kappa B translocates to the nucleus with concomitant degradation of I kappa B. We show that cold-induced depolymerization of microtubules also leads to I kappa B degradation and activation of NF-kappa B. However, the activated factor remains in the cytoplasm and translocates to the nucleus only upon warming to 37 degrees C, thus revealing two distinct steps in NF-kappa B activation. These findings establish a new role for NF-kappa B in sensing changes in the state of the cytoskeleton and converting them to changes in gene activity.

NF-kappa B activation by ultraviolet light not dependent on a nuclear signal.

Exposure of mammalian cells to radiation triggers the ultraviolet (UV) response, which includes activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B). This was postulated to occur by induction of a nuclear signaling cascade by damaged DNA. Recently, induction of AP-1 by UV was shown to be mediated by a pathway involving Src tyrosine kinases and the Ha-Ras small guanosine triphosphate-binding protein, proteins located at the plasma membrane. It is demonstrated here that the same pathway mediates induction of NF-kappa B by UV. Because inactive NF-kappa B is stored in the cytosol, analysis of its activation directly tests the involvement of a nuclear-initiated signaling cascade. Enucleated cells are fully responsive to UV both in NF-kappa B induction and in activation of another key signaling event. Therefore, the UV response does not require a signal generated in the nucleus and is likely to be initiated at or near the plasma membrane.

p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction.

The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role.

Molecular cloning and characterization of a novel Rel/NF-kappa B family member displaying structural and functional homology to NF-kappa B p50/p105.

The NF-kappa B transcription factor has been implicated in the inducible expression of many genes, including inflammatory, immune, and acute-phase response genes. NF-kappa B consists of two subunits, 50K and 65K polypeptides. The genes encoding p50 and p65 have sequence similarities with the c-rel proto-oncogene and the Drosophila maternal effect gene dorsal. We describe the cloning and characterization of a novel rel-related gene encoding a 98K product that shares extensive homology with the p105 precursor of the NF-kappa B p50 protein, containing both a Rel homology and SWI6/ankyrin repeat domain. We demonstrate that p98 is proteolytically processed in vivo to generate a 55K polypeptide, which binds to kappa B sites. p55 is capable of forming heterocomplexes with other Rel/NF-kappa B family members, which can bind to kappa B motifs in vitro, and stimulate transcription of reporter genes containing these cis-elements in vivo. The identification of a homolog for NF-kappa B p50/p105, termed p55/p98, gives further support to the idea that NF-kappa B is a collection of structurally related complexes of which contribute to the pleiotropic regulatory processes originally assigned to NF-kappa B.

Isolation, localization, and physical mapping of a highly polymorphic locus on human chromosome 11q13.

A highly polymorphic repetitive sequence, D11S533, was isolated by oligonucleotide hybridization from an arrayed chromosome 11q-specific cosmid library. The DNA sequence of this element was determined and found to consist of a repetitive degenerate hexanucleotide sequence [T(Pu)T(Pu)T(Pu)]n extending over 438 bp. Southern blot analysis demonstrated that this element is relatively unique in the human genome. This sequence can be detected by amplification using the polymerase chain reaction (PCR) with oligonucleotide primers complementary to unique sequences flanking the repetitive element. This sequence displays a high degree of polymorphism, and analysis of 15 individuals demonstrated at least 10 alleles ranging in size from 300 to 900 bp. Fluorescence in situ hybridization was used to localize this sequence to 11q13 (FLpter 0.60 +/- 0.02). Pulsed-field gel electrophoresis and the isolation of yeast artificial chromosomes established the long-range physical map surrounding the locus. Because various alleles of this polymorphic sequence can be easily detected by PCR amplification, this probe has potential usefulness in genetic linkage mapping as well as identity testing.

Relationship of c-myc gene copy number and gene expression: cellular effects of elevated c-myc protein.

The relationship between gene copy number and expression and cellular consequences of elevated levels of c-myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c-myc gene. HC-8 and LC-5 recombinant cells carry approximately 800 and 50 copies of c-myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c-myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC-8 cells produced approximately tenfold more c-myc protein than heated LC-5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c-myc expression was observed following heat induction, even though the amounts of genomic c-myc were relatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c-myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c-myc protein in HC-8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c-myc protein are incompatible with cell survival.

Generation of clonal DNA templates for in vitro transcription without plasmid purification.

WE present a rapid procedure based on the polymerase chain reaction for generation of double-stranded DNA templates suitable for in vitro transcription by T3 or T7 RNA polymerase. DNA fragments cloned into a phage promoter vector are amplified together with a flanking promoter to provide functional templates. Extension of oligonucleotide primer molecules harboring an RNA polymerase promoter sequence at their 5'-end allows positioning of the transcription start site within the insert. The procedure generates large amounts of linear transcription template without need to isolate and purify plasmid DNA from bacterial cells.