Fully automatic segmentation of the mitral leaflets in 3D transesophageal echocardiographic images using multi-atlas joint label fusion and deformable medial modeling.

Comprehensive visual and quantitative analysis of in vivo human mitral valve morphology is central to the diagnosis and surgical treatment of mitral valve disease. Real-time 3D transesophageal echocardiography (3D TEE) is a practical, highly informative imaging modality for examining the mitral valve in a clinical setting. To facilitate visual and quantitative 3D TEE image analysis, we describe a fully automated method for segmenting the mitral leaflets in 3D TEE image data. The algorithm integrates complementary probabilistic segmentation and shape modeling techniques (multi-atlas joint label fusion and deformable modeling with continuous medial representation) to automatically generate 3D geometric models of the mitral leaflets from 3D TEE image data. These models are unique in that they establish a shape-based coordinate system on the valves of different subjects and represent the leaflets volumetrically, as structures with locally varying thickness. In this work, expert image analysis is the gold standard for evaluating automatic segmentation. Without any user interaction, we demonstrate that the automatic segmentation method accurately captures patient-specific leaflet geometry at both systole and diastole in 3D TEE data acquired from a mixed population of subjects with normal valve morphology and mitral valve disease.

Over-parameterised, uncertain 'mathematical marionettes' - how can we best use catchment water quality models? An example of an 80-year catchment-scale nutrient balance.

The Integrated Catchment Model of Nitrogen (INCA-N) was applied to the River Lambourn, a Chalk river-system in southern England. The model's abilities to simulate the long-term trend and seasonal patterns in observed stream water nitrate concentrations from 1920 to 2003 were tested. This is the first time a semi-distributed, daily time-step model has been applied to simulate such a long time period and then used to calculate detailed catchment nutrient budgets which span the conversion of pasture to arable during the late 1930s and 1940s. Thus, this work goes beyond source apportionment and looks to demonstrate how such simulations can be used to assess the state of the catchment and develop an understanding of system behaviour. The mass-balance results from 1921, 1922, 1991, 2001 and 2002 are presented and those for 1991 are compared to other modelled and literature values of loads associated with nitrogen soil processes and export. The variations highlighted the problem of comparing modelled fluxes with point measurements but proved useful for identifying the most poorly understood inputs and processes thereby providing an assessment of input data and model structural uncertainty. The modelled terrestrial and instream mass-balances also highlight the importance of the hydrological conditions in pollutant transport. Between 1922 and 2002, increased inputs of nitrogen from fertiliser, livestock and deposition have altered the nitrogen balance with a shift from possible reduction in soil fertility but little environmental impact in 1922, to a situation of nitrogen accumulation in the soil, groundwater and instream biota in 2002. In 1922 and 2002 it was estimated that approximately 2 and 18 kg N ha(-1) yr(-1) respectively were exported from the land to the stream. The utility of the approach and further considerations for the best use of models are discussed.

An evaluation of the relationship between specialist training in cardiology and implementation of evidence-based care of patients following acute myocardial infarction.

AIMS: Large clinical trials have provided evidence of prognostically beneficial treatment strategies for patients with acute myocardial infarction. However, the implementation of this evidence into routine clinical practice is suboptimal. We hypothesised that the speciality of the attending physician (cardiologist or not) would affect the use of evidence-based strategies. METHODS: Over a 3-month period (1st September to 30th November 1995), 3684 consecutive potential cases of acute myocardial infarction (AMI) in 20 adjacent hospitals in the Yorkshire Region were identified from coronary care registers, clinical coding and biochemistry records of cardiac enzyme assay requests. There were 2153 consecutive cases of AMI identified, of which 1643 patients were alive at discharge. We compared the admission use of aspirin and thrombolysis, and the use of aspirin, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors and statins at discharge between cardiologists and other physicians. RESULTS: AMI patients under the care of cardiologists are more likely to receive aspirin and thrombolysis on the day of their event and to be prescribed aspirin, beta-blockers and statins on discharge. After correction for contraindications to their use, the above findings were broadly confirmed. DISCUSSION: Cardiologists are more likely than general physicians to use evidence-based treatment strategies recognised to improve AMI patient outcome. It is likely that this will translate into a reduction of mortality or other hard endpoints in patient outcomes.

Identification of evolutionarily conserved promoter elements and amino acids required for function of the C. elegans beta-catenin homolog BAR-1.

beta-catenins are conserved transcription factors regulated posttranslationally by Wnt signaling. bar-1 encodes a Caenorhabditis elegans beta-catenin acting in multiple Wnt-mediated processes, including cell fate specification by vulval precursor cells (VPCs) and migration of the Q(L) neuroblast progeny. We took two approaches to extend our knowledge of bar-1 function. First, we undertook a bar-1 promoter analysis using transcriptional GFP reporter fusions and found that bar-1 expression is regulated in specific cells at the transcriptional level. We identified promoter elements necessary for bar-1 expression in several cell types, including a 321-bp element sufficient for expression in ventral cord neurons (VCNs) and a 1.1-kb element sufficient for expression in the developing vulva and adult seam cells. Expression of bar-1 from the 321-bp element rescued the Uncoordinated (Unc) phenotype of bar-1 mutants, but not the vulval phenotype, suggesting that a Wnt pathway may act in ventral cord neurons to mediate proper locomotion. By comparison of the 1.1-kb element to homologous sequences from Caenorhabditis briggsae, we identified evolutionarily conserved sequences necessary for expression in vulval or seam cells. Second, we analyzed 24 mutations in bar-1 and identified several residues required for BAR-1 activity in C. elegans. By phylogenetic comparison, we found that most of these residues are conserved and may identify amino acids necessary for beta-catenin function in all species.

Identifying acute myocardial infarction: effects on treatment and mortality, and implications for National Service Framework audit.

BACKGROUND: The National Service Framework (NSF) for Coronary Heart Disease requires annual clinical audit of the care of patients with myocardial infarction, with little guidance on how to achieve these standards and monitor practice. AIM: To assess which method of identification of acute myocardial infarction (AMI) cases is most suitable for NSF audit, and to determine the effect of the definition of AMI on the assessment of quality of care. DESIGN: Observational study. METHODS: Over a 3-month period, 2153 consecutive patients from 20 hospitals across the Yorkshire region, with confirmed AMI, were identified from coronary care registers, biochemistry records and hospital coding systems. The sensitivity and positive predictive value of AMI patient identification using clinical coding, biochemistry and coronary care registers were compared to a 'gold standard' (the combination of all three methods). RESULTS: Of 3685 possible cases of AMI singled out by one or more methods, 2153 patients were identified as having a final diagnosis of AMI. Hospital coding revealed 1668 (77.5%) cases, with a demographic profile similar to that of the total cohort. Secondary preventative measures required for inclusion in NSF were also of broadly similar distribution. The sensitivities and positive predictive values for patient identification were substantially less in the cohorts identified through biochemistry and coronary care unit register. Patients fulfilling WHO criteria (n=1391) had a 30-day mortality of 15.9%, vs. 24.2% for the total cohort. DISCUSSION: Hospital coding misses a substantial proportion (22.5%) of AMI cases, but without any apparent systematic bias, and thus provides a suitably representative and robust basis for NSF-related audit. Better still would be the routine use of multiple methods of case identification.

Poor prognosis of patients presenting with symptomatic myocardial infarction but without chest pain.

OBJECTIVE: To describe the clinical features, prognosis, and treatment of patients presenting with atypical forms of acute myocardial infarction. DESIGN: Consecutive cases of possible acute myocardial infarction were sought from coronary care registers, biochemistry records, and hospital management systems. Case notes were reviewed and predefined epidemiological and clinical variables were abstracted. SETTING: 20 adjacent hospitals in the former Yorkshire region. PATIENTS: 3684 consecutive cases of possible acute myocardial infarction admitted in a three month period were identified, of whom 2096 had a first episode of confirmed acute myocardial infarction. RESULTS: 20.2% of all patients admitted with an eventual diagnosis of acute myocardial infarction presented with symptoms other than chest pain. Compared with the group presenting with chest pain, these patients were older (76.6 v 69.1 years, p < 0.001), were more often women (54.6% v 35.3%, p < 0.001), and were more likely to have a history of heart failure (18.6% v 6.9%, p < 0.001). They had a higher 30 and 365 day mortality (49.2% and 61.0%, respectively) compared with patients presenting with chest pain (17.9% and 26.2%). In a Cox regression analysis the hazard ratio for presentation without chest pain was 1.60 (95% confidence interval 1.30 to 1.97) (p < 0.001) adjusted for age, heart rate, blood pressure, left ventricular impairment, and infarction with ST segment elevation as covariates. Importantly, they were also less likely to receive treatments with a proven ability to improve prognosis. CONCLUSIONS: Atypical presentation of myocardial infarction without chest pain is common and associated with increased mortality. This may result in part from a failure to use beneficial treatment strategies.

Results of the expanded program on immunization in the Macedonian refugee camps.

OBJECTIVES: This report summarizes the results of the Macedonian refugee camps' Expanded Program on Immunization. METHODS: Government agencies and nongovernmental organizations implemented an immunization program consisting of 3 mass vaccination campaigns in each of the 7 camps. Before the second mass campaign, weekly immunization clinics were initiated in each camp. Children younger than 48 months were immunized against 8 antigens according to a schedule established by the Macedonian Ministry of Health. RESULTS: Immunization coverage rates in the second campaign were 91% in Cegrane and 73% in Brazda. Coverage rates of the weekly clinics averaged 93%. CONCLUSIONS: Initiating an expanded immunization program in the absence of a stable population is problematic.

Use of cumulative mortality data in patients with acute myocardial infarction for early detection of variation in clinical practice: observational study.

OBJECTIVES: Use of cumulative mortality adjusted for case mix in patients with acute myocardial infarction for early detection of variation in clinical practice. DESIGN: Observational study. SETTING: 20 hospitals across the former Yorkshire region. PARTICIPANTS: All 2153 consecutive patients with confirmed acute myocardial infarction identified during three months. MAIN OUTCOME MEASURES: Variable life-adjusted displays showing cumulative differences between observed and expected mortality of patients; expected mortality calculated from risk model based on admission characteristics of age, heart rate, and systolic blood pressure. RESULTS: The performance of two individual hospitals over three months was examined as an example. One, the smallest district hospital in the region, had a series of 30 consecutive patients but had five more deaths than predicted. The variable life-adjusted display showed minimal variation from that predicted for the first 15 patients followed by a run of unexpectedly high mortality. The second example was the main tertiary referral centre for the region, which admitted 188 consecutive patients. The display showed a period of apparently poor performance followed by substantial improvement, where the plot rose steadily from a cumulative net lives saved of -4 to 7. These variations in patient outcome are unlikely to have been revealed during conventional audit practice. CONCLUSIONS: Variable life-adjusted display has been integrated into surgical care as a graphical display of risk-adjusted survival for individual surgeons or centres. In combination with a simple risk model, it may have a role in monitoring performance and outcome in patients with acute myocardial infarction.

A simple benchmark for evaluating quality of care of patients following acute myocardial infarction.

OBJECTIVE: To develop a simple risk model as a basis for evaluating care of patients admitted with acute myocardial infarction. METHODS: From coronary care registers, biochemistry records and hospital management systems, 2153 consecutive patients with confirmed acute myocardial infarction were identified. With 30 day all cause mortality as the end point, a multivariable logistic regression model of risk was constructed and validated in independent patient cohorts. The areas under receiver operating characteristic curves were calculated as an assessment of sensitivity and specificity. The model was reapplied to a number of commonly studied subgroups for further assessment of robustness. RESULTS: A three variable model was developed based on age, heart rate, and systolic blood pressure on admission. This produced an individual probability of death by 30 days (P(30)) where P(30) = 1/(1 + exp(-L(30))) and L(30) = -5.624 + (0.085 x age) + (0.014 x heart rate) - (0.022 x systolic blood pressure). The areas under the receiver operating characteristic curves for the reference and test cohorts were 0.79 (95% CI 0.76 to 0.82) and 0.76 (95% CI 0.72 to 0.79), respectively. To aid application of the model to routine clinical audit, a normogram relating observed mortality and sample size to the likelihood of a significant deviation from the expected 30 day mortality rate was constructed. CONCLUSIONS: This risk model is simple, reproducible, and permits quality of care of acute myocardial infarction patients to be reliably evaluated both within and between centres.

Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Delta mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Transcriptional activation by Gcn4p involves independent interactions with the SWI/SNF complex and the SRB/mediator.

Mutations in three subunits of the SWI/SNF complex and in the Med2p subunit of the SRB/mediator of pol II holoenzyme impaired Gcn4p-activated transcription of HIS3 without reducing Gcn4p-independent transcription of this gene. Recombinant Gcn4p interacted with SWI/SNF and SRB/mediator subunits in cell extracts in a manner dependent on the same hydrophobic clusters in the Gcn4p activation domain; however, higher concentrations of Gcn4p were required for binding to SWI/SNF versus SRB/mediator subunits. In addition, SRB/mediator and SWI/SNF subunits did not coimmunopreciptate from the extracts. These findings, together with the fact that Gcn4p specifically interacted with purified SWI/SNF, strongly suggest that Gcn4p independently recruits SWI/SNF and holoenzyme to its target promoters in the course of activating transcription.

Restraining infarct expansion preserves left ventricular geometry and function after acute anteroapical infarction.

BACKGROUND: Expansion of an acute myocardial infarction predicts progressive left ventricular (LV) dilatation, functional deterioration, and early death. This study tests the hypothesis that restraining expansion of an acute infarction preserves LV geometry and resting function. METHODS AND RESULTS: In 23 sheep, snares were placed around the distal left anterior descending and second diagonal coronary arteries. In 12 sheep, infarct deformation was prevented by Marlex mesh placed over the anticipated myocardial infarct. Snared arteries were occluded 10 to 14 days later. Serial hemodynamic measurements and transdiaphragmatic quantitative echocardiograms were obtained up to 8 weeks after anteroapical infarction of 0.23 of LV mass. In sheep with mesh, circulatory hemodynamics, stroke work, and end-systolic elastance return to preinfarction values 1 week after infarction and do not change subsequently. Ventricular volumes and ejection fraction do not change after the first week postinfarction. Control animals develop large anteroapical ventricular aneurysms, increasing LV dilatation, and progressive deterioration in circulatory hemodynamics and ventricular function. At week 8, differences in LV end-diastolic pressure, cardiac output, end-diastolic and end-systolic volumes, ejection fraction, stroke work, and end-systolic elastance are significant (P<0.01) between groups. CONCLUSIONS: Preventing expansion of acute myocardial infarctions preserves LV geometry and function.

yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex.

We obtained a recessive insertion mutation in the gene encoding yeast TBP-associated factor yTAFII61/68 that impairs Gcn4p-independent and Gcn4p-activated HIS3 transcription. This mutation also reduces transcription of seven other class II genes, thus indicating a broad role for this yTAFII in RNA polymerase II transcription. The Gcn4p activation domain interacts with multiple components of the SAGA complex in cell extracts, including the yTAFII proteins associated with SAGA, but not with two yTAFIIs restricted to TFIID. The taf61-1 mutation impairs binding of Gcn4p to SAGA/yTAFII subunits but not to components of holoenzyme mediator. Our results provide strong evidence that recruitment of SAGA, in addition to holoenzyme, is crucial for activation by Gcn4p in vivo and that yTAFII61 plays a key role in this process.

Infarct size and location determine development of mitral regurgitation in the sheep model.

OBJECTIVE: This study tests the hypothesis that neither small nor large myocardial infarctions that include the anterior papillary muscle produce mitral regurgitation in sheep. METHODS: Coronary arterial anatomy to the anterior left ventricle and papillary muscle was determined by dye injection in 41 sheep hearts and by triphenyl tetrazolium chloride in 13. Development of acute or chronic mitral regurgitation and changes in left ventricular dimensions were studied by use of transdiaphragmatic echocardiography in 21 sheep after infarction of 24% and 33% of the anterior left ventricular mass. These data were compared with previous data from large and small posterior left ventricular infarctions. RESULTS: Ligation of two diagonal arteries infarcts 24% of the left ventricular mass and 82% of the anterior papillary muscle. Ligation of both diagonals and the first circumflex branch infarcts 33% of the left ventricle and all of the anterior papillary muscle. Neither infarction causes mitral regurgitation, although left ventricular cavity dimensions increase significantly at end systole. After the smaller infarction, the left ventricular cavity enlarges 150% over 8 weeks without mitral regurgitation. CONCLUSIONS: In sheep small and large infarctions of the anterior wall that include the anterior papillary muscle do not produce either acute or chronic mitral regurgitation despite left ventricular dilatation. In contrast large posterior infarctions produce immediate mitral regurgitation owing to asymmetric annular dilatation and discoordination of papillary muscle relationships to the valve. After small posterior infarctions that include the posterior papillary muscle, mitral regurgitation develops because of annular and ventricular dilatation during remodeling.

The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex.

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

Papillary muscle discoordination rather than increased annular area facilitates mitral regurgitation after acute posterior myocardial infarction.

BACKGROUND: Acute posterior myocardial infarction that produces immediate mitral regurgitation alters the mitral annulus and its spatial relationship with both papillary muscles. The precise deformations that cause valve insufficiency are not understood and impair efforts to repair the valve. METHODS AND RESULTS: In six Dorsett hybrid sheep, sonomicrometry transducers were placed around the mitral annulus (6) and at the tips and bases of both papillary muscles (4). Two weeks later, three branches of the circumflex coronary artery were occluded to infarct approximately 32% of the posterior left ventricle. This infarction produced acute 2 to 3+ mitral regurgitation in all animals, as determined by color flow Doppler velocity mapping. Before and after infarction, distance measurements between sonomicrometry transducers were used to produce the three-dimensional coordinates of each transducer every 5 ms. After infarction, the area of the annulus increased only 9.2+/-6.3% at end systole (ES). In addition, the normal shortening of the posterior papillary muscle was obliterated to allow its tip to move 1.4+/-0.6 mm closer to the centroid of the annulus at ES. After infarction, the anterior papillary muscle continued to shorten normally, but at ES, its tip and base were 0.9+/-0.7 mm and 1.3+/-0.7 mm farther from the centroid, respectively. CONCLUSIONS: These deformations tend to produce a relative prolapse of leaflet tissue attached to the posterior papillary muscle and restriction of leaflet tissue attached to the anterior papillary muscle. This papillary muscle discoordination with minimal annular dilatation distorts leaflet coaptation sufficiently to produce severe mitral regurgitation.

Distortions of the mitral valve in acute ischemic mitral regurgitation.

BACKGROUND: In the absence of papillary muscle rupture, the precise deformations that cause acute postinfarction mitral valve regurgitation are not understood and impair reparative efforts. METHODS: In 6 Dorsett hybrid sheep, sonomicrometry transducers were placed around the mitral annulus (n = 6) and at the tips and bases of both papillary muscles (n = 4). Later, specific circumflex coronary arteries were occluded to infarct approximately 32% of the posterior left ventricle and produce acute 2 to 3+ mitral regurgitation. Before and after infarction, distance measurements between sonomicrometry transducers produced three-dimensional coordinates of each transducer every 5 ms. RESULTS: After infarction, the annulus dilated asymmetrically orthogonal to the line of leaflet coaptation, but the annular area increased only 9.2% +/- 6.3% (p = 0.02). At end-systole, posterior papillary muscle length increased 2.3 +/- 0.9 mm (p = 0.005); the posterior papillary muscle tip moved closer to the annular plane and centroid, and the anterior papillary muscle tip moved away. CONCLUSIONS: Small deformations in mitral valvular spatial geometry after large posterior infarctions are sufficient to produce moderate to severe mitral regurgitation. The most important changes are asymmetric annular dilatation, prolapse of leaflet tissue tethered by the posterior papillary muscle, and restriction of leaflet tissue attached to the anterior papillary muscle.

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR.

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.

Identification of seven hydrophobic clusters in GCN4 making redundant contributions to transcriptional activation.

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.