Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.

The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13. By positional cloning, we have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients. On mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it ranges from 7 to 17 repeats. Gonadal instability in SCA7 is greater than that observed in any of the seven known neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal transmissions. SCA7 is the first such disorder in which the degenerative process also affects the retina.

RING-dependent tumor suppression and G2/M arrest induced by the TRC8 hereditary kidney cancer gene.

TRC8/RNF139 and von Hippel-Lindau (VHL) both encode E3 ubiquitin (Ub) ligases mutated in clear-cell renal carcinomas (ccRCC). VHL, inactivated in nearly 70% of ccRCCs, is a tumor suppressor encoding the targeting subunit for a Ub ligase complex that downregulates hypoxia-inducible factor-alpha. TRC8/RNF139 is a putative tumor suppressor containing a sterol-sensing domain and a RING-H2 motif essential for Ub ligase activity. Here we report that human kidney cells are growth inhibited by TRC8. Inhibition is manifested by G2/M arrest, decreased DNA synthesis and increased apoptosis and is dependent upon the Ub ligase activity of the RING domain. Tumor formation in a nude mouse model is inhibited by TRC8 in a RING-dependent manner. Expression of TRC8 represses genes involved in cholesterol and fatty acid biosynthesis that are transcriptionally regulated by the sterol response element binding proteins (SREBPs). Expression of activated SREBP-1a partially restores the growth of TRC8-inhibited cells. These data suggest that TRC8 modulation of SREBP activity comprises a novel regulatory link between growth control and the cholesterol/lipid homeostasis pathway.

Myxoid liposarcoma with t(12;16) (q13;p11) contains site-specific differences in methylation patterns surrounding a zinc-finger gene mapped to the breakpoint region on chromosome 12.

The q13 to q15 region of human chromosome 12 is frequently and consistently rearranged in malignant and benign adipose tissue tumors as well as benign tumors of smooth muscle and salivary glands. A reciprocal translocation, (12;16) (q13;p11), is characteristic of the myxoid subtype of liposarcoma, whereas translocations within 12q13-14 are frequently observed in benign lipomas. We are using pulsed-field gel electrophoresis to study the 12q13-q14 region in order to detect and clone the respective translocation breakpoints in these tumors. The locus GLI, which encodes a zinc-finger protein, has been mapped to the same region as the myxoid liposarcoma breakpoint. Pulsed-field analysis of myxoid liposarcoma and lipoma DNA has allowed us to construct a 600-kilobase physical map surrounding the GLI locus, which shows that breakpoints in both types of tumor are outside this region. However, myxoid liposarcoma DNA samples contained altered restriction fragments detectable with GLI probes that were highly specific and reproducible from case to case. These altered fragments are due to highly specific and reproducible methylation differences that are unique to myxoid liposarcoma DNA. These methylation changes may prove to be useful clinically as a diagnostic tool to differentiate subtypes of liposarcoma.

Homozygous deletions of human chromosome 3p in lung tumors.

Cytogenetic and loss of heterozygosity (LOH) studies have demonstrated that deletions of chromosome 3p occur at a high frequency in all forms of lung cancer. To clarify the role of 3p in lung tumorigenesis and to more precisely identify targets for positional cloning efforts, we have performed 3p deletion analyses (microsatellite and fluorescence in situ hybridization) in a series of lung cancer cell lines and uncultured tumor samples. Importantly, we identified homozygous deletions in four uncultured tumors and one cell line. Homozygous deletions were found in three squamous tumors within a region of 3p21 which had previously been described only in cell lines, a 1-2-megabase homozygous deletion in a small cell tumor at 3p12, and a 3p14.2 homozygous deletion in a non-small cell lung carcinoma cell line. The detection of homozygous deletions affecting these multiple regions in uncultured tumor cells substantiates the belief (previously based on deletions found only in tumor cell lines) that these sites contain important tumor suppressor genes. Along with previously reported homozygous deletions in a distal portion of 3p21.3, we now have evidence for four separate regions of 3p which undergo homozygous deletions in either uncultured lung tumors or cell lines.

Chromosomal duplication accompanies allelic loss in non-small cell lung carcinoma.

Hemizygous deletion in the short (p) arm of chromosome 3 is a common finding in non-small cell lung carcinoma (NSCLC) and is postulated to be a crucial early change in lung tumorigenesis. Yet one of the most frequent nuclear abnormalities in both NSCLC and premalignant bronchial epithelium is increase in chromosomal copy number. Deletion and duplication have not been assessed in the same tumor set by both molecular and cytogenetic methods to determine whether allelic loss correlates with chromosomal duplication in the same tumor cell populations. It is also not established what biological mechanisms might lead to allelic deletion and chromosomal duplication. We have investigated changes in the copy number of chromosome 3 in touch preparations of 38 NSCLCs (19 adenocarcinomas and 19 squamous cell carcinomas) using dual-target, dual-color fluorescence in situ hybridization (FISH) assays. Chromosome 3 centromere probe was matched with a 3p14.2 probe [intron 4 of the fragile histidine triad (FHIT) gene] and a 3p21.31 probe (HSemaIV gene). We then correlated FISH results with results of molecular analyses for allelic losses at loci in the regions to which the FISH probes mapped in 20 of these cases. Although various combinations of FISH abnormalities were sometimes detected within the same specimens, individual cases could be classified according to the predominant FISH pattern, usually with one abnormality present in >60% of tumor cells. Chromosomal duplication, indicated by the presence of more than two centromeric signals, was the most frequent abnormality observed by FISH and was accompanied by loss of specific sequences on 3p in approximately one-half of the specimens in which it was observed. The most frequent abnormality observed by molecular analysis was loss of heterozygosity (LOH) in both of the chromosomal regions tested and was demonstrated in 83% of cases with chromosomal duplication. We conclude that LOH may occur in the presence of chromosomal duplication, suggesting that the duplicated chromosome is homozygous. Our findings imply that LOH occurs before chromosomal duplication during lung carcinogenesis.

Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.

Cytogenetic and molecular studies have implied the presence of tumor suppressor genes (TSGs) on chromosome 9p that are critical in the development of lung and other cancers. The p16/CDKN2 gene, a cyclin dependent kinase inhibitor, is a well-defined TSG on 9p21. Although the frequency of mutations in the p16/CDKN2 gene has been detected in approximately 30% of non-small cell lung cancer, loss of heterozygosity on 9p has been observed in greater than 70% of non-small cell lung cancers. These and other deletion mapping studies have suggested the existence of additional TSGs on 9p. This study examined chromosome 9p for TSG loci by analyzing 23 squamous cell carcinomas of the lung with 21 microsatellite markers. Loss of heterozygosity was detected in all of the tumors, and homozygous deletions of the p16/ CDKN2 locus were observed in 6 of the 23 tumors (26%). In addition, a novel region of homozygous deletion was detected in six tumors (26%) at D9S126, approximately 2.5 cM proximal to p16/CDKN2. A single tumor contained a homozygous deletion at both the p16/CDKN2 locus and the D9S126 locus. The possibility of homozygous loss was confirmed by multiplex PCR using both the D9S126 marker and a chromosome 9p control marker. Fluorescence in situ hybridization analysis with P1 and cosmid probes containing D9S126 also confirmed these data. The minimum region of homozygous deletion was determined by testing markers immediately proximal and distal to the D9S126 region. The data identify a homozygous loss on the short arm of chromosome 9 suggesting the presence of a novel TSG locus, proximal to p16/CDKN2 and located between D9S265 and D9S259.

The use of nutritional supplements in dressage and eventing horses.

The aim of the study was to determine which types of nutritional supplements were used in dressage and eventing horses, and the reasons that owners used supplements. An online questionnaire was distributed through British Eventing and Dressage websites, to collect data on demographics of owners and their horses, supplements used and their opinion on health and performance problems. Data were evaluated using descriptive analysis, Sign and Fisher's exact tests for quantitative data, and categorisation of qualitative data. In total, 599 responses met the inclusion criteria (441 dressage and 158 eventing horse owners). Participants had 26.4 (3-60) (mean (range)) years of riding experience, owned 1.2 (0-10) horses and used 2 (0-12) supplements in their highest performing horse. The main health and performance issues identified for dressage were 'energy/behaviour', 'lameness' and 'back and muscle problems'. The main issues for eventing were 'stamina and fitness levels',' lameness' and 'energy/behaviour'. The main reasons for using supplements in their highest performing horse were 'joints and mobility', and 'behaviour' for dressage, and 'electrolytes', and 'joints and mobility' for eventing. Lameness and behavioural problems were significant concerns within both disciplines. There was incongruence between owners' opinions of problems within their discipline and their reasons for using supplements.

Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin.

Loss of chromosome 3p is a critical event in the pathogenesis of lung cancer. Overlapping homozygous 3p21.3 deletions in lung cancer cell lines involving GNAI2 were characterized and found to involve a region of genomic instability. A new widely expressed Semaphorin, H.SemaIV, was isolated from the GNAI2 deletion region. Reduced H.SemaIV expression allowed identification of additional cell lines with submicroscopic or larger deletions of the locus which occurred in a heterogeneous manner. We also demonstrate the presence of a distinct 3p21.3 homozygous deletion region, adjacent to the DNA mismatch repair gene, hMLH1, and identified deletions in direct tumors. This appears to represent one of the first demonstrations of homozygous deletions affecting 3p in direct lung tumors.

DEF-3(g16/NY-LU-12), an RNA binding protein from the 3p21.3 homozygous deletion region in SCLC.

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins.

PTEN/MMAC1 mutations identified in small cell, but not in non-small cell lung cancers.

A putative tumor suppressor, PTEN/MMAC1 gene at 10q23 was recently identified and found to be mutated in many different human tumors. To determine the role of the PTEN/MMAC1 gene in lung cancer, we screened 34 small cell lung cancer (SCLC) cell lines, 10 SCLC tumors, 13 non-small cell lung cancer (NSCLC) cell lines and 10 NSCLC tumors using Denaturing HPLC (DHPLC) and direct sequencing methods. In SCLC, six (18%) of the cell lines and one of the primary tumor samples (10%) showed alterations of the PTEN/MMAC1 gene including point mutations, small fragment deletions, and homozygous deletions. All of the point mutations and small fragment deletions were observed in hemizygously deleted cell lines. In contrast to SCLC, none of the NSCLC tumors or cell lines had mutations in the PTEN/MMAC1 gene. These data indicate that PTEN/MMAC1 mutations contribute to the pathogenesis and neoplastic evolution in SCLC but not in NSCLC.

High-throughput tissue microarray analysis used to evaluate biology and prognostic significance of the E-cadherin pathway in non-small-cell lung cancer.

PURPOSE: E-cadherin (E-cad) and its associated intracellular molecules, catenins, are critical for intercellular epithelial adhesion and are often expressed in non-small-cell lung carcinomas (NSCLCs). We constructed tissue microarrays (TMAs) to investigate the expression of cadherins and catenins and their prognostic significance in NSCLC. PATIENTS AND METHODS: Tumor tissue samples from 193 patients with stages I to III NSCLC were obtained from the University of Colorado Cancer Center and Johns Hopkins Medical Institutions. Viable tumor was sampled in triplicate for the TMAs, and slides were stained by immunohistochemistry with antibodies against E-cad, N-cadherin, alpha (alpha)-, beta (beta)-, and gamma (gamma)-catenin, p120, p27, and adenomatous polyposis coli (APC) gene product. Clinical data were collected by the tumor registries. Patients were followed for a median period of 51 months (range, 18 to 100 months). RESULTS: Absent or severely reduced membranous expression for E-cad, alpha-, beta-, and gamma-catenin, and p120 were observed in 10%, 17%, 8%, 31%, and 61% of the cases, respectively. Tumor cell dedifferentiation correlated with reduced expression for E-cad, beta-catenin, gamma-catenin, and p120 in squamous cell carcinomas but not in adenocarcinomas. There was an inverse correlation between nodal metastasis and expression of E-cad and gamma-catenin. Besides the traditional clinical prognostic variables, E-cad and alpha-, beta-, and gamma-catenin expression were of positive prognostic value in univariate survival analyses. In multivariate analysis, E-cad expression was the only independent prognostic factor for survival in addition to age, node status, tumor status, and pathologic surgical margins. CONCLUSION: Reduced expression of E-cad and catenins is associated with tumor cell dedifferentiation, local invasion, regional metastasis, and reduced survival in NSCLC. E-cad is an independent prognostic factor for NSCLC survival.

Transcription initiation sites of the leucine operons of Salmonella typhimurium and Escherichia coli.

Evidence for a transcription attenuation site downstream from the leu promoter was obtained by transcription experiments in vitro. Most transcription initiated in vitro from leuP is terminated prematurely, resulting in the synthesis of a 160 nucleotide leader RNA. We define here the point at which transcription is initiated in vitro and in vivo and demonstrate that the site of premature termination is between the promoter and the first structural gene (leuA). Additional nucleotide sequences are presented that extend the known sequence 200 base-pairs upstream and 300 base-pairs downstream from leuP. The location of the promoter-proximal end of cistron leuA was deduced by comparing nucleotide sequence data with the sequence of the ten amino acids at the N-terminus of alpha-isopropylmalate synthase. To facilitate the isolation of quantities of material for sequencing experiments, the enzyme was isolated from a plasmid-containing strain, CV605, grown under conditions of leucine limitation. Under such conditions, about 20% of the total soluble protein of strain CV605 is alpha-isopropylmalate synthase and another 20% is beta-isopropylmalate dehydrogenase (leuB product).

Molecular cloning of alpha-amylase genes from Drosophila melanogaster. I. Clone isolation by use of a mouse probe.

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.

Molecular cloning of alpha-amylase genes from Drosophila melanogaster. II. Clone organization and verification.

Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.

Hox expression in AML identifies a distinct subset of patients with intermediate cytogenetics.

We previously reported that favorable and poor prognostic chromosomal rearrangements in acute myeloid leukemia (AML) were associated with distinct levels of HOX expression. We have now analyzed HOX expression in 50 independent adult AML patients (median age=62 years), together with FLT3 and FLT3-ligand mRNA levels, and FLT3 mutation determination. By cluster analysis, we could divide AMLs into cases with low, intermediate and high HOX expression. Cases with high expression were uniquely restricted to a subset of AMLs with intermediate cytogenetics (P=0.0174). This subset has significantly higher levels of FLT3 expression and appears to have an increase of FLT3 mutations (44%), while CEBPalpha mutations were infrequent (6%). FLT3 mRNA levels were correlated with the expression of multiple HOX genes, whereas FLT3 mutations were correlated with HOXB3. In some cases, FLT3 was expressed at levels equivalent to GAPDH in the absence of genomic amplification. We propose that high HOX expression may be characteristically associated with a distinct biologic subset of AML. The apparent global upregulation of HOX expression could be due to growth-factor signaling or, alternatively, these patterns may reflect a particular stage of differentiation of the leukemic cells.

Quantitative HOX expression in chromosomally defined subsets of acute myelogenous leukemia.

We used a degenerate RT-PCR screen and subsequent real-time quantitative RT-PCR assays to examine the expression of HOX and TALE-family genes in 34 cases of chromosomally defined AML for which outcome data were available. AMLs with favorable cytogenetic features were associated with low overall HOX gene expression whereas poor prognostic cases had high levels. Characteristically, multiple HOXA family members including HOXA3-HOXA10 were jointly overexpressed in conjunction with HOXB3, HOXB6, MEIS1 and PBX3. Higher levels of expression were also observed in the FAB subtype, AML-M1. Spearmann correlation coefficients indicated that the expression levels for many of these genes were highly inter-related. While we did not detect any significant correlations between HOX expression and complete response rates or age in this limited set of patients, there was a significant correlation between event-free survival and HOXA7 with a trend toward significance for HoxA9, HoxA4 and HoxA5. While patients with elevated HOX expression did worse, there were notable exceptions. Thus, although HOX overexpression and clinical resistance to chemotherapy often coincide, they are not inextricably linked. Our results indicate that quantitative HOX analysis has the potential to add new information to the management of patients with AML, especially where characteristic chromosomal alterations are lacking.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer.

Lung carcinogenesis is assumed to be a multistep process, but detailed understanding of the sequential morphological and molecular changes preceding invasive lung cancer remains elusive. To better understand early lung carcinogenesis, we initiated a program of fluorescence bronchoscopy in smokers at high risk for lung cancer. In the bronchial biopsies from these subjects, we observed a unique lesion consisting of capillary blood vessels closely juxtaposed to and projecting into metaplastic or dysplastic squamous bronchial epithelium, angiogenic squamous dysplasia (ASD). Serial sections of the capillary projections confirmed that they represent intramucosal capillary loops. Microvessel density in ASD was elevated in comparison to normal mucosa (P = 0.0003) but not in comparison to other forms of hyperplasia or dysplasia. ASD thus represents a qualitatively distinct form of angiogenesis in which there is architectural rearrangement of the capillary microvasculature. Genetic analysis of surface epithelium in a random subset of lesions revealed loss of heterozygosity at chromosome 3p in 53% of ASD lesions. No confirmed p53 mutations were identified. Compared with normal epithelium, proliferative activity was markedly elevated in ASD lesions. ASD occurred in 54 of 158 (34%) high-risk smokers without carcinoma and in 6 of 10 patients with squamous carcinoma who underwent fluorescence bronchoscopy. One early-stage invasive carcinoma was noteworthy for the occurrence of ASD juxtaposed to invasive tumor. Seventy-seven (59%) of the ASD lesions were detected by abnormal fluorescence alone. Twenty bronchial sites (11 patients) were rebiopsied 1 year after the initial diagnosis. At nine (45%) of these sites, the lesion was found to persist. The lesion was not present in biopsies from 16 normal nonsmoker control subjects. The presence of this lesion in high-risk smokers suggests that aberrant patterns of microvascularization may occur at an early stage of bronchial carcinogenesis.

A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium.

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.