A comprehensive review of MR imaging changes following radiosurgery to 500 brain metastases.

BACKGROUND AND PURPOSE: Stereotactic radiosurgery is known to control 85%-95% of intracranial metastatic lesions during a median survival of 6-8 months. However, with the advent of newer systemic cancer therapies, survival is improving; this change mandates a longitudinal quantitative analysis of the radiographic response of brain metastases to radiosurgery. MATERIALS AND METHODS: MR imaging of 516 metastases in 120 patients treated with GK-SRS from June 2006 to December 2009 was retrospectively reviewed. Lesion volume at initial treatment and each follow-up was calculated by using the following formula: length × width × height / 2. Volume changes were correlated with patient demographics, histopathology, and radiation treatment variables. RESULTS: Thirty-two percent of lesions increased in volume following radiosurgery. Clinically, this translated into 54% of patients having ≥1 of their lesions increase in size. This increase begins at 6 weeks and can last beyond 15 months' post-SRS. Male sex (P = .002), mean voxel dose <37 Gy (P = .009), and initial treatment volume >500 mm(3) (P < .001) are associated with posttreatment increases in tumor size. Median survival following radiosurgery was 9.5 months for patients with all lesions exhibiting stable/decreased volumes, >18.4 months for patients with all lesions exhibiting increased volumes, and 16.4 months for patients with mixed lesional responses. CONCLUSIONS: Most metastatic lesions are stable or smaller in size during the first 36 months post-SRS. However, a transient increase in volume is seen in approximately one-third of lesions. Sex, treatment dose, initial lesion size, and histopathology all correlate with variations in lesion volume post-SRS. The longer the patient survives, the more likely an increase in lesion size will be seen on follow-up imaging.

Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa.

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-microm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm(-1), differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.

Ventriculopleural shunt obstruction and positive-pressure ventilation. Case report.

The authors report an unusual case of a patient with low-pressure hydrocephalus and a ventriculopleural shunt, in whom routine respiratory management performed using positive-pressure ventilation caused shunt obstruction and coma. While the patient received positive-pressure ventilation with external cerebrospinal fluid (CSF) drainage at subatmospheric pressure, the ventricles returned to normal size and the coma rapidly reversed. After the authors' recognition of the effect of positive-pressure ventilation on intrapleural pressure and ventriculopleural shunt function, and the subsequent removal of positive-pressure ventilation, CSF flow through the shunt resumed and the patient's coma resolved.

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

cDNA cloning, chromosomal localization, and expression analysis of human BEHAB/brevican, a brain specific proteoglycan regulated during cortical development and in glioma.

BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.

A xylem-specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress.

Angiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant decrease in lignin in the cell walls of tension-stressed tissues. To investigate the molecular basis of this tree stress response, we cloned a full-length cellulose synthase (PtCesA) cDNA from developing xylem of aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts in various aspen tissues, and PtCesA gene promoter-beta-glucuronidase (GUS) fusion analysis in transgenic tobacco, demonstrated conclusively that PtCesA expression is confined to developing xylem cells during normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in xylem and was induced in developing phloem fibers undergoing tension stress, but was turned off in tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for PtCesA in cellulose biosynthesis in both tension-stressed and normal tissues in aspen, and that the on/off control of PtCesA expression may be a part of a signaling mechanism triggering a stress-related compensatory deposition of cellulose and lignin that is crucial to growth and development in trees.

5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms.

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.

Toward more rational prediction of outcome in patients with high-grade subarachnoid hemorrhage.

OBJECTIVE: Accurate outcome prediction after high-grade subarachnoid hemorrhage remains imprecise. Several clinical grading scales are in common use, but the timing of grading and changes in grade after admission have not been carefully evaluated. We hypothesized that these latter factors could have a significant impact on outcome prediction. METHODS: Fifty-six consecutive patients with altered mental status after subarachnoid hemorrhage, who were managed at a single institution, were studied retrospectively. On the basis of prospectively assessed elements of the clinical examination, each patient was graded at admission, at best before treatment, at worst before treatment, immediately before treatment, and at best within 24 hours after treatment of the aneurysm using the Glasgow Coma Scale (GCS), the World Federation of Neurological Surgeons (WFNS) scale, and the Hunt and Hess scale. Outcome at 6 months was determined using a modification of the Glasgow Outcome Scale validated against the Karnofsky scale. All grades and clinical and radiographic data collected were compared among good and poor outcome groups. Multivariate analyses were then performed to determine which grading scale, which time of grading, and which other factors were correlated with and contributed significantly to outcome prediction. RESULTS: A good outcome was achieved in 24 (43%) of 56 patients. Our study also had a 32% mortality rate. With the Hunt and Hess scale, only the worst pretreatment grade was significantly correlated with outcome. However, with the GCS and the WFNS scale, grading at all pretreatment times was significantly correlated with outcome, although outcome was best predicted before treatment, regardless of the scale used, if grading was performed at the patient's clinical worst. Multivariate analysis revealed that the best predictor of outcome was WFNS grade at clinical worst before treatment. Used alone, a WFNS Grade 3 at worst pretreatment predicted a 75% favorable outcome, and a WFNS Grade 5 at worst pretreatment predicted an 87% poor outcome. No significant correlation was found between direction or magnitude of change in grade and outcome. Age was found to be significantly correlated with outcome, but it was only an independent factor in outcome prediction when used in conjunction with the Hunt and Hess scale and not with the WFNS scale and the GCS. CONCLUSION: Timing of grading is an important factor in outcome prediction that needs to be standardized. This study suggests that the patient's worst clinical grade is most predictive of outcome, especially when the patient is assessed using the WFNS scale or the GCS.

Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine.

Two types of structurally distinct O-methyltransferases mediate the methylation of hydroxylated monomeric lignin precursors in angiosperms. Caffeate 3-O-methyltransferase (COMT; EC 2.1.1.68) methylates the free acids and caffeoyl CoA 3-O-methyltransferase (CCoAOMT; EC 2.1.1.104) methylates coenzyme A esters. Recently, we reported a novel hydroxycinnamic acid/hydroxycinnamoyl CoA ester O-methyltransferase (AEOMT) from loblolly pine differentiating xylem that was capable of methylating both acid and ester precursors with similar efficiency. In order to determine the possible existence and role of CCoAOMT in lignin biosynthesis in gymnosperms, a 1.3 kb CCoAOMT cDNA was isolated from loblolly pine that showed 79-82% amino acid sequence identity with many angiosperm CCoAOMTs. The recombinant CCoAOMT expressed in Escherichia coli exhibited a significant methylating activity with hydroxycinnamoyl CoA esters whereas activity with hydroxycinnamic acids was insignificant. Moreover, 3.2 times higher catalytic efficiency for methylating caffeoyl CoA over 5-hydroxyferuloyl CoA was observed which could serve as a driving force towards synthesis of guaiacyl lignin. The secondary xylem-specific expression of CCoAOMT was demonstrated using RNA blot analysis, western blot analysis, and O-methyltransferase enzyme assays. In addition, Southern blot analysis indicated that CCoAOMT may exist as a single-copy gene in loblolly pine genome. The transgenic tobacco plants carrying loblolly pine CCoAOMT promoter-GUS fusion localized the site of GUS activity at the secondary xylem tissues. These data suggest that CCoAOMT, in addition to AEOMT, plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine.

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees.

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.

Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms.

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.

Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases.

Plant S-adenosyl-L-methionine-dependent methyltransferases (SAM-Mtases) are the key enzymes in phenylpropanoid, flavonoid and many other metabolic pathways of biotechnological importance. Here we compiled the amino acid sequences of 56 SAM-Mtases from different plants and performed a computer analysis for the conserved sequence motifs that could possibly act as SAM-binding domains. To date, genes or cDNAs encoding at least ten distinct groups of SAM-Mtases that utilize SAM and a variety of substrates have been reported from higher plants. Three amino acid sequence motifs are conserved in most of these SAM-Mtases. In addition, many conserved domains have been discovered in each group of O-methyltransferases (OMTs) that methylate specific substrates and may act as sites for substrate specificity in each enzyme. Finally, a diagrammatic representation of the relationship between different OMTs is presented. These SAM-Mtase sequence signatures will be useful in the identification of SAM-Mtase motifs in the hitherto unidentified proteins as well as for designing primers in the isolation of new SAM-Mtases from plants.

Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides).

4-Coumarate:CoA ligases (4CLs, EC 6.2.1.12) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants. So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics. However, we have discovered two functionally and structurally distinct 4CL genes, Pt4CL1 and Pt4CL2 (63% protein sequence identity), that are differentially expressed in aspen (Populus tremuloides). The Escherichia coli-expressed and purified Pt4CL1 and Pt4CL2 proteins exhibited highly divergent substrate preference as well as specificity that reveal the association of Pt4CL1 with the biosynthesis of guaiacyl-syringyl lignin and the involvement of Pt4CL2 with other phenylpropanoid formation. Northern hybridization analysis demonstrated that Pt4CL1 mRNA is specifically expressed in lignifying xylem tissues and Pt4CL2 mRNA is specifically expressed in epidermal layers in the stem and the leaf, consistent with the promoter activities of Pt4CL1 and Pt4CL2 genes based on the heterologous promoter-beta-glucouronidase fusion analysis. Thus, the expression of Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen; Pt4CL1 is devoted to lignin biosynthesis in developing xylem tissues, whereas Pt4CL2 is involved in the biosynthesis of other phenolics, such as flavonoids, in epidermal cells.

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants.

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and ß-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l-1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Molecular cloning of 4-coumarate:coenzyme A ligase in loblolly pine and the roles of this enzyme in the biosynthesis of lignin in compression wood.

Two genomic sequences encoding 4-coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) in loblolly pine (Pinus taeda L.) were cloned. Both sequences contained three introns and four exons with identical coding sequences predicting 537 amino acids. Two of the three introns in these two clones were different both in sequence and in length. Sequences of both 4CL clones were found in all nine megagametophyte DNAs tested, providing genetic evidence that these two 4CL genomic sequences are nonallelic genes. Our analyses suggest that there are at least two distinct, intron-containing 4CL genes, at least one of which is transcribed into 4CL mRNA in developing xylem tissue of loblolly pine. The levels of 4CL gene transcription in xylem were influenced by compressional stress, resulting in an elevated 4CL enzyme activity with 4-coumaric acid. 4CL enzyme activity with ferulic acid remained unchanged, whereas with caffeic acid it was significantly inhibited. Exogenously applied trans-cinnamic acid in the protein extracts from normal wood xylem caused inhibition of 4CL activity toward caffeic acid similar to that under compressional stress. The implications of this cinnamic acid-modulated effect on 4CL enzyme activities toward different substrates in regulating monolignol synthesis in xylem under compressional stress are discussed.

Context sequences of translation initiation codon in plants.

In this survey of 5074 plant genes for their AUG context sequences, purines are present at the -3 and +4 positions in about 80% of the sequences. Although this observation is similar to the vertebrate consensus sequence, the number of plant mRNAs with purines at the -3 position is lower and at the +4 position is higher than reported for vertebrate mRNAs. Higher plants have an AC-rich consensus sequence. caA(A/C)aAUGGCg as a context of translation initiator codon. Between the two major groups of angiosperms, the context of the AUG codon in dicot mRNAs is aaA(A/C)aAUGGCu which is similar to the higher-plant consensus but monocot mRNAs have c(a/c)(A/G)(A/C)cAUGGCG as a consensus which exhibits an overall similarity with the vertebrate consensus. The experimental evidence regarding the importance of the AUG context in plants is discussed.

3' cycle-labeled oligonucleotides with predictable length for primer extension and transgene analysis.

Efficient labeling of short oligos at their 3'-ends was achieved through polymerase chain reaction. The length of cycled-labeled oligos can be accurately predicted by omitting one or more dNTPs in the labeling step. Thus, labeled oligos can be simply column-purified, eliminating the need for tedious gel purification. We demonstrated the effectiveness of this technique in determining the transcription start site of a given gene and in transgene analysis to differentiate the transcript of an endogenous gene from that of an introduced homologous gene. This technique could be widely extended to other molecular biology applications in which labeled oligos are employed.