RESUMEN
Inherited disorders of surfactant metabolism are manifested in neonatal period as a severe respiratory failure not responding to exogenous surfactant administration. We illustrate the case of a term newborn with respiratory failure because of compound heterozygous mutation in adenosine triphosphate-binding cassette transporter A3 (ABCA3)-in exon 24 M1227R and in exon 29 Ins1510fs/ter1519. These mutations of ABCA3 have not been described yet and expand the group of lethal ABCA3 variants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Insuficiencia Respiratoria/genética , Análisis Mutacional de ADN , Resultado Fatal , Heterogeneidad Genética , Variación Genética , Ventilación de Alta Frecuencia , Humanos , Recién Nacido , Masculino , Insuficiencia Respiratoria/terapia , Nacimiento a TérminoRESUMEN
Interferon (IFN)-lambda1, -2 and -3 (also designated as interleukin (IL)-29, IL-28alpha and IL-28beta) represent a new subfamily within the class II cytokine family. They show type I IFN-like antiviral and cytostatic activities in affected cells forming the basis for IFN-lambda1 therapy currently under development for hepatitis C infection. However, many aspects of IFN-lambdas are still unknown. This study aimed at identifying the target cells of IFN-lambdas within the immune system and the skin. Among skin cell populations, keratinocytes and melanocytes, but not fibroblasts, endothelial cells or subcutaneous adipocytes turned out to be targets. In contrast to these target cells, blood immune cell populations did not clearly respond to even high concentrations of these cytokines, despite an IFN-lambda receptor expression. Interestingly, immune cells expressed high levels of a short IFN-lambda receptor splice variant (sIFN-lambdaR1/sIL-28R1). Its characterization revealed a secreted, glycosylated protein that binds IFN-lambda1 with a moderate affinity (K(D) 73 nM) and was able to inhibit IFN-lambda1 effects. Our study suggests that IFN-lambda therapy should be suited for patients with verrucae, melanomas and non-melanoma skin cancers, apart from patients with viral hepatitis, and would not be accompanied by immune-mediated complications known from type I IFN application.
Asunto(s)
Interferones/inmunología , Leucocitos/inmunología , Receptores de Interferón/inmunología , Secuencia de Aminoácidos , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Queratinocitos/inmunología , Melanocitos/inmunología , Datos de Secuencia Molecular , Receptores de Interferón/química , Receptores de Interferón/genéticaRESUMEN
Bovine leukaemia virus (BLV) is an oncogenic retrovirus that causes B-cell lymphocytosis and in the terminal stage of the disease lymphosarcoma. The comparison of the previously published BLV provirus sequence from Belgium, Australia and Japan showed that the protease gene (prt) of the Australian and the Japanese isolate contain a nucleotide deletion when compared to the Belgian isolate. Because all these proviruses were isolated from tumour tissue, the prt gene of functionally active and infectious proviruses from peripheral blood leucocytes (PBLs) of BLV-infected cattle and from BLV-infected fetal lamb kidney cells were sequenced. The only variations between these sequences and the Belgian isolate consist of nucleotide substitutions. The delection of one nucleotide of the prt gene of the Japanese and the Australian BLV tumour isolate caused a changed reading frame and a premature translational stop. It was shown that the Japanese provirus is non-infectious in transfected cell culture and in injected sheep. To analyse the impact of the prt mutation on viral protein expression and infectivity, the prt region of the Japanese provirus was exchanged with the prt region from the Belgian provirus. The resulting pBLVprtbelg was infectious in transfected cells and enabled the expression of gag and gag-precursor proteins. One sheep was injected with this mutated provirus and became positive in BLV-PCR, but no seroconversion was developed. The prt mutation of the Japanese tumour isolates was shown to be responsible for the loss of infectivity and changed viral expression. These results and the occurrence of this mutation in only two isolates from lymphosarcoma indicate a possible relation between the prt mutation and the induction of cell transformation.
Asunto(s)
Leucosis Bovina Enzoótica/virología , Eliminación de Gen , Regulación Viral de la Expresión Génica , Virus de la Leucemia Bovina/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , ADN Viral/aislamiento & purificación , Endopeptidasas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Biosíntesis de Proteínas , Sistemas de Lectura/genética , Alineación de Secuencia/veterinaria , Ovinos , Proteínas Virales/químicaRESUMEN
The alveolar surfactant is a prime target of reactive oxygen species present in air. Alveolar surfactant is supplemented with vitamin E during its assembly in type II pneumocytes. However, it is unknown which of the lipoproteins supply type II pneumocytes with vitamin E. The measurement of the uptake kinetics indicates that HDL might be the primary source of the vitamin E uptake by type II pneumocytes. Vitamin E depletion of rats caused an increase of vitamin E uptake by isolated type II pneumocytes from HDL but not from LDL or VLDL. We demonstrated that type II pneumocytes express the scavenger receptor class B type 1 (SR-B1), an HDL-specific receptor. Vitamin E depletion caused an increased expression of SR-B1 by a post-transcriptional mechanism. The increased vitamin E uptake from HDL and the increased expression of the SR-B1 were reversed by refeeding the vitamin. We propose that HDL is the primary source of vitamin E for type II pneumocytes. The rate of uptake of vitamin E by this cell type might be regulated by the expression of SR-B1.
Asunto(s)
Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Vitamina E/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Cinética , Lipoproteínas/metabolismo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores Depuradores , Receptores Depuradores de Clase B , Deficiencia de Vitamina E/metabolismoRESUMEN
Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 +/- 1.8 nM; maximum velocity = 62.7 +/- 5.8 pmol. min(-1). 5 x 10(5) pneumocytes(-1)) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0 degrees C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo-N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261-268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.
Asunto(s)
Pulmón/metabolismo , Glicoproteínas de Membrana/fisiología , Transportadores de Anión Orgánico , Ácido Palmítico/farmacocinética , Secuencia de Aminoácidos/genética , Animales , Compuestos Bicíclicos con Puentes/farmacología , Antígenos CD36 , Sinergismo Farmacológico , Pulmón/citología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ácidos Oléicos/farmacología , Ácido Palmítico/antagonistas & inhibidores , Floretina/farmacología , Ratas , Ratas Wistar , Succinimidas/farmacologíaRESUMEN
A typical infection with bovine leukemia virus (BLV) induces a permanent antibody (Ab) response with high titers against BLV-antigens. In the last few years atypical courses of infection with low or transient BLV-Ab-titers or even lack of any detectable BLV-Ab-titers in animals with BLV-provirus integration have been described. This makes it difficult to eliminate BLV infection from herds using serological assays only. Whether or not polymerase chain reaction (PCR) is a useful tool to complement serological Ab-assays in BLV-eradication in herds was clarified in three ways: (i) different DNA-quick-preparations of blood were examined in nested PCR, (ii) cows of a BLV infected herd that was involved in a national eradication program were investigated for 6 months und (iii) BLV-provirus-variants occurring in this herd were differentiated. The results show, that even by using PCR it was not possible to detect all infected animals all the time and that eradication of BLV from this herd was not completed in this short time. The PCR is useful for the investigation of herds and more sensitive than ELISA. PCR using LTR-primers (34 positive cattle) was more sensitive than PCR with env-primers (30 positive cattle). Using PCR 34 BLV infected cattle were detected of which only 21 reacted in ELISA. Restriction enzyme analysis or sequence analysis of PCR-amplificates allowed the detection of virus variants and conclusions about the way of infection. PCR should be used for BLV-eradication in cattle herds with low BLV-incidence, for the investigation of new outbreaks or tumor cases in long term BLV free herds and for investigation of breeding cattle.
Asunto(s)
ADN Viral/química , Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Bovinos , ADN Viral/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Apart from dipalmitoyl phosphatidylcholine, cholesterol is the most abundant surfactant lipid. About 90 to 99% of cholesterol of the alveolar surfactant is derived from serum lipoproteins. The aim of this study was to identify the lipoprotein which preferentially supplements type II pneumocytes with cholesterol destined for surfactant production. Ultrastructural investigations revealed that type II pneumocytes bind and take up HDL, LDL and VLDL. Binding and uptake of VLDL occurred even in the presence of excess LDL indicating that, besides LDL receptors, type II pneumocytes express additional binding sites for VLDL. Type II pneumocytes in primary culture are able to take up cholesterol added in the form of HDL, LDL and VLDL. Cholesterol uptake was lowest from HDL and highest from VLDL. The maximal velocity of cholesterol uptake from VLDL was more than three times that of cholesterol uptake from LDL. The half-maximal saturation of cholesterol uptake from VLDL was nearly half that of LDL. From these kinetic data and the distribution of free cholesterol among the serum lipoproteins, we calculated that the cholesterol uptake from VLDL is more than three times that of cholesterol uptake from LDL. In double-labeling experiments type II pneumocytes secreted palmitic acid-labeled phospholipids together with labeled free cholesterol taken up from lipoproteins. The secretion rates of both phospholipids and free cholesterol were stimulated to nearly the same extent by isoproterenol. From our results we conclude that type II pneumocytes interact specifically with HDL, LDL and VLDL. Cholesterol taken up in the form of the individual lipoproteins shows no difference in its availability for the formation of cholesterol ester and surfactant by type II pneumocytes in vitro. Based on the kinetic studies, it appears that VLDL is the major gateway through which cholesterol is provided to satisfy the cholesterol requirements of type II pneumocytes for the synthesis of surfactant.
Asunto(s)
Lipoproteínas/metabolismo , Pulmón/citología , Pulmón/metabolismo , Animales , Adhesión Celular/fisiología , Colesterol/metabolismo , Colesterol/farmacocinética , Ésteres del Colesterol/metabolismo , Oro Coloide/metabolismo , Histocitoquímica , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacocinética , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacocinética , Lipoproteínas VLDL/metabolismo , Lipoproteínas VLDL/farmacocinética , Pulmón/química , Masculino , Fosfolípidos/metabolismo , Unión Proteica , Surfactantes Pulmonares/biosíntesis , Ratas , Ratas Wistar , Tritio/metabolismoRESUMEN
Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.
Asunto(s)
Enfermedades de los Bovinos/virología , Leucosis Bovina Enzoótica/virología , Virus de la Leucemia Bovina/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Variación Genética , Datos de Secuencia MolecularRESUMEN
The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.
Asunto(s)
Apoproteínas/metabolismo , Endocitosis , Pulmón/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Células Cultivadas , Metabolismo de los Lípidos , Masculino , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Ratas , Receptores de Superficie Celular/metabolismoRESUMEN
An Aspergillus niger endopolygalacturonase (EC 3.2.1.15) cDNA was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein into the growth medium was efficiently directed by the fungal leader sequence, and processing occurred at the same site as in Aspergillus. The expression level was significantly enhanced by using a "short" version of the yeast ADHI promoter. An additional increase in the yield of heterologous protein was due to a higher plasmid stability and a rise in plasmid copy number. This was achieved by deleting most of the bacterial sequences from the expression vector. The yeast-derived enzyme showed the same enzymatic and biochemical properties as the fungal polygalacturonase, such as substrate specificity, pH and temperature optima and pI value. The yeast-derived enzyme, however, showed a higher degree of glycosylation and exhibited a more pronounced temperature stability than the fungal enzyme.
Asunto(s)
Aspergillus niger/enzimología , Poligalacturonasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Alcohol Deshidrogenasa/genética , Secuencia de Bases , Fermentación , Glicosilación , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
By introducing synthetic oligonucleotides into a lacZ-yeast expression vector a set of 47 plasmids (out of 64 possible) was generated, differing only in the three bases immediately upstream of the AUG initiation codon of the Escherichia coli lacZ gene. Expression of the beta-galactosidase fusion protein encoded by the different plasmids was determined in Saccharomyces cerevisiae by immunogel electrophoresis. Among the clones tested we found a factor 3 difference in expression. A slight nucleotide preference was found in positions -3(A > G > C = U) and -2 (G > C = U > A). The choice of the nucleotide at position -1 immediately 5' of the AUG did not effect translation efficiency. Increasing homology to the yeast consensus sequence (AAAAAAAUGUCU) was not concomitant with an increased translation efficiency. Our results indicate that the choice of nucleotides immediately preceding the initiation codon in yeast does not dramatically influence translation efficiency, as in prokaryotes or higher eukaryotes.
Asunto(s)
Codón , Escherichia coli/genética , Operón Lac , Nucleótidos/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , PlásmidosRESUMEN
A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis. A 5.3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.
Asunto(s)
Codón/genética , Regulación Fúngica de la Expresión Génica , Operón Lac , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Escherichia coli/genética , Inmunoelectroforesis , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Transformación Genética , beta-Galactosidasa/biosíntesisRESUMEN
The basic replicon of the endogenous Methylomonas clara plasmid pBE-2 and its derivatives was defined to a region of 2.7 kb by in vivo deletions and conjugative transfer experiments using Escherichia coli-M. clara hybrid plasmids. Origin activity was found to be confined to a maximal length of 1.3 kb. The origin consists of two fragments which can be separated more than 4 kb by the integration of foreign DNA fragments without loss of function. A fragment having a maximum size of 2.1 kb supports in trans replication initiation at the origin. In addition, two incompatibility determinants were revealed, one localized in the origin fragment and the other outside the origin. Incompatibility between two basic replicons of the natural M. clara plasmids can be overcome by the integration of one of them in the compatible IncP plasmid R68-Kms. No homology was found between the plasmid basic replicon and the chromosomal DNA of M. clara.
Asunto(s)
Methylococcaceae/genética , Plásmidos , Replicón , Deleción Cromosómica , Cromosomas Bacterianos , Conjugación Genética , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Escherichia coli/genética , Etidio/farmacología , Methylococcaceae/efectos de los fármacos , Mutación , Hibridación de Ácido Nucleico , Mapeo RestrictivoRESUMEN
In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.
Asunto(s)
Codón , Escherichia coli/genética , Genes , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero , Secuencia de Bases , Vectores Genéticos , ARN Mensajero/genética , ARN de Transferencia/genéticaRESUMEN
Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.
Asunto(s)
Escherichia coli/fisiología , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Regulación de la Expresión Génica , Genes Bacterianos , Genes Virales , Operón Lac , Conformación de Ácido Nucleico , Relación Estructura-Actividad , Proteínas Virales/genéticaRESUMEN
We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.
Asunto(s)
Codón , Escherichia coli/genética , Genes Bacterianos , Operón Lac , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero , Secuencia de Bases , Sitios de Unión , Mutación , Biosíntesis de Proteínas , ARN Bacteriano/genética , ARN Mensajero/genética , Ribosomas/metabolismo , beta-Galactosidasa/biosíntesisRESUMEN
A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon. The lacZ translation initiation signals [Shine-Dalgarno (SD) sequence and AUG codon] were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI. With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage Q beta A2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins. The introduction of N-terminal amino acids (aa) in beta-galactosidase (beta-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme. Therefore an immunoprecipitation method was used to measure the amount of fusion protein. It was found that these amounts varied strongly from one construction to another. Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones. No strict correlation could be found between the level of lac mRNA and beta-gal production. Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.