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PubMed1991

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.

Acyl Carrier Protein↗
PubMed2003

Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage.

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither beta-glucuronidase nor beta-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT-rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae.

Bacterial Proteins↗
PubMed2003

Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery.

The single-chain antibody (scFv) made by recombinant DNA technology is one of the most useful tools for basic research and clinical applications. To develop a novel targeted gene delivery method, we engineered the scFv gene for the antibody against human epidermal growth factor (EGF) receptor by connecting with DNA sequences for various oligopeptides with negative or positive charges. The resulting recombinant genes encoding artificial scFv with negative or positive tails were expressed in Escherichia coli and yeast Pichia pastris. In E. coli, all the scFv with negatively charged tails were expressed but mainly as an insoluble form, whereas those with positively charged tails were barely expressed. In yeast P. pastris, all the scFv with negatively charged tails were efficiently expressed and secreted into the culture medium. Addition of high salt into the yeast culture increased their secretion. Purification procedure was established for the scFv with the longest negatively charged tail (D4S x 5), yielding 5 mg/l with a purity of over 95%. The scFv-D4S x 5 was designated as a recombinant immunoporter, which was then mixed with plasmid DNA and polyethylenimine (PEI). The resulting DNA/PEI/immunoporter complex (designated recombinant immunogene) exhibited efficient gene delivery to EGF receptor overexpressing A431 tumor cells.

Antibodies↗
PubMed1993

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants.

A microbial expression system for the study of the cysteine protease papain has been developed as a more useful alternative to the insect cell/baculovirus expression system we have previously used. A synthetic papain precursor (propapain) gene was expressed in the yeast Saccharomyces cerevisiae under the control of the alpha-factor promoter. Efficient expression required fusion of the propapain sequence with the yeast alpha-factor prepro region and a yeast host cell defective in the synthesis of vacuolar proteases. Surprisingly, the glycosylated form of the inactive papain precursor is not secreted, but accumulates within the yeast cell. Complete conversion of the intracellular zymogen into active mature papain could be achieved in vitro. Purified recombinant papain produced by the yeast system has kinetic characteristics similar to those of the natural enzyme. An advantage of the yeast expression system over the baculovirus/insect cell system is that we can perform mutagenesis and screening of papain mutants very efficiently. We have set up a 'one-tube' screening procedure for the simultaneous characterization of numerous mutants of the papain precursor. Yeast cells are grown and lysed in microtiter plate wells and the released papain precursor is then activated to mature papain. This assay allows easy discrimination between proteins with close to wild type properties and proteins that are not functional. We have applied this assay to investigate the spectrum of amino acids which are tolerated at Asn175 of papain using two independently derived libraries of mutants at this position. Many amino acid substitutions at this position are not accepted; only the reintroduction of Asn restored normal function.

Amino Acid Sequence↗
PubMed1985

Expression of a synthetic human growth hormone gene in yeast.

A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 10(6) molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.

Alkaline Phosphatase↗
PubMed2003

Structural features affecting variant surface glycoprotein expression in Trypanosoma brucei.

The glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of Trypanosoma brucei is the most abundant GPI-anchored protein expressed on any cell, and is an essential virulence factor. To determine what structural features affect efficient expression of VSG, we made a series of mutations in two VSGs. Inserting 18 amino acids, between the amino- and carboxy-terminal domains, reduced the expression of VSG 221 to about 3% of the wild-type level. When this insertion was combined with deletion of the single carboxy-terminal subdomain, expression was reduced a further three-fold. In VSG 117, which contains two carboxy-terminal subdomains, point mutation of the intervening N-glycosylation site reduced expression about 15-fold. Deleting the most carboxy-terminal subdomain and intervening region, including the N-glycosylation site, reduced expression to 15-20% of wild type VSG, and deletion of both subdomains reduced expression to <1%. Despite their low abundance, all VSG mutants were GPI anchored on the cell surface. Our results suggest that, for a protein to be efficiently displayed on the surface of bloodstream-form T. brucei, it is essential that it contains the conserved structural motifs of a T. brucei VSG. Serum resistance-associated protein (SRA), which confers human infectivity on T. brucei, strongly resembles a VSG deletion mutant. Expression of three epitope-tagged versions of SRA in T. brucei conferred total resistance to human serum. SRA possesses a canonical GPI signal sequence, but we were unable to obtain unequivocal evidence for the presence of a GPI anchor. SRA was not released during osmotic lysis, indicating that it is not GPI anchored on the cell surface.

Amino Acid Sequence↗
PubMed1988

Structure and expression of the ompB operon, the regulatory locus for the outer membrane porin regulon in Salmonella typhimurium LT-2.

The ompB operon of Salmonella typhimurium encodes a positive transcriptional regulator OmpR and an inner membrane protein EnvZ. Both proteins are needed for the proper expression of the outer membrane proteins OmpC and OmpF. We have determined the nucleotide sequence of the ompB locus and its adjacent regions. A comparison between the S. typhimurium and Escherichia coli sequences revealed that the ompB locus is highly conserved. The sequence data also showed that ompR and envZ form an operon, where the coding regions overlap by four base-pairs. Utilizing ompR-lacZ and envZ-lacZ gene fusions, the translational levels of expression of these two genes were measured, showing that ompR is considerably more efficiently expressed than envZ. Analysis of ompR frameshift mutations showed that translation of envZ is almost totally dependent on the translation of the upstream gene ompR. The mechanism of this translational coupling appears to be a reinitiation of the ribosome at the overlapping region of the two genes. The characteristics of the OmpR and EnvZ proteins were in agreement with the known functions and cellular locations of these proteins. OmpR was found to contain a putative DNA binding site, while EnvZ contained two hydrophobic stretches typical of transmembrane regions. Both OmpR and EnvZ show extensive homologies with many proteins from a number of different origins, all of which function in pairs and through which environmental signals modulate gene expression. Hence, the tightly coupled synthesis of these proteins seems to be essential in eliciting a proper response in the transmembrane regulation of gene expression.

Amino Acid Sequence↗
PubMed1991

Protection of the hypertrophied myocardium by crystalloid cardioplegia.

Patients with left ventricular hypertrophy (LVH) have a worse outcome after cardiac surgery than those without hypertrophy. We studied protection of hearts with LVH in an isolated rat heart model using multidose, cold, oxygenated cardioplegia. LVH was produced by banding the abdominal aorta in young rats. Six weeks after banding, this produced a 31% increase in the left ventricular dry weight/body weight ratio compared to two age-matched control groups comprising sham-operated and nonoperated animals. The recovery of cardiac output after arrest was higher in LVH (82 +/- 4% of prearrest) than in sham-operated (69 +/- 4%) or nonoperated (66 +/- 3%) control groups. The improved functional recovery in LVH occurred although there were no differences among the groups in myocardial adenosine triphosphate (ATP) and phosphocreatine (PCr) prior to arrest, at the end of arrest, or after reperfusion. Glycogen levels were also similar among the three groups prior to arrest and after reperfusion but were highest in LVH after arrest. Myocardial oxygen consumption (MVO2) and efficiency, expressed as cardiac output/MVO2, were similar among the groups prior to arrest. Myocardial efficiency after reperfusion declined in all groups but was best preserved in LVH. We also compared the sensitivity of hypertrophied and control hearts to the deleterious effects of calcium in cardioplegia. Calcium in the cardioplegia increased myocardial lactate production during arrest in a dose-related fashion and depressed myocardial levels of ATP, PCr, and glycogen at end arrest in all groups. Cardiac output recovery was also depressed by calcium but was still best in LVH. We conclude that the hypertrophied myocardium is well protected by standard cardioplegia and that calcium in cardioplegia does not preferentially depress recovery in LVH.

Animals↗
PubMed1989

Inefficient expression in rat cells of transforming gene of BK virus with 68-bp tandem repeats in the viral promoter-enhancer.

Large-plaque BK virus (wt-501), which has three 68-bp tandem repeats in the viral promoter-enhancer, is not transforming for rat cells, whereas the minute-plaque mutant (dl-504) with only one 68-bp element is. We tested the BKV wt-501 and dl-504 promoter-enhancers for their ability to activate the heterologous and homologous genes in rat 3Y1 cells. The 501 promoter-enhancer activated expression of the chloramphenicol acetyltransferase gene and the neomycin-resistance gene more efficiently than the 504 promoter-enhancer. The 501 enhancer could not allow the efficient expression of BK virus T-antigen gene, whereas the 504 enhancer could. The wt-BKV enhancer with three 68-bp repeats seemed to function positively on the heterologous genes but negatively on the homologous transforming gene in rat cells. The sequence causing the suppressive effects by duplication was confined within the 29-bp segment containing two SV40 enhancer core homologies in the 68-bp element. The results show that inability of wt-BKV to transform rat cells is ascribable to inefficient T-antigen gene expression caused by the duplication in the enhancer and by the surrounding or downstream DNA sequence.

Animals↗
PubMed1992

TcR-alpha mRNA accumulation does not dictate cell surface TcR/CD3 expression.

The TcR-alpha chain is the last subunit of the TcR/CD3 complex to be expressed during thymic ontogeny. Since the presence of all subunits are required for efficient expression of this complex on the cell surface, this has lead to the hypothesis that the TcR-alpha chain is the limiting subunit which controls cell surface TcR/CD3 expression during thymocyte differentiation. We have examined this issue using a T-lymphoma cell clone, RS4.2, which has a CD4- CD8- Thyl+ J11d+ IL2R+ phenotype, identical with immature thymocytes which have the capacity to generate all major T cell subsets. The RS4.2 cell clone accumulates abundant amounts of TcR-beta, CD3-gamma, -delta, -epsilon and -zeta transcripts, but expresses trace levels of TcR-alpha transcripts and cell surface TcR/CD3. TcR-alpha mRNA levels can be dramatically augmented in RS4.2 cells by three distinct mechanisms: in response to treatment with either phorbol myristate acetate (PMA), calcium ionophore (A23187), or cycloheximide (CHX). However, the expression of TcR/CD3 on the cell surface fails to be increased by any of these agents. In fact, PMA induces a rapid down-regulation of cell surface TcR/CD3 expression. In contrast, these agents trigger an increase in cell surface IL2R expression which coincides with augmented IL2R-alpha mRNA levels. Thus, in RS4.2 cells, IL2R surface expression is controlled by transcript levels, while TcR/CD3 surface expression is regulated by post-transcriptional events, independent of TcR-alpha mRNA accumulation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2000

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes.

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.

Cell Nucleus↗
PubMedDate not supplied

Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide.

We have recently described a new method for attaching padlock oligonucleotides to supercoiled plasmid DNA at specific sequences. A variant of this method has been developed in order to allow the coupling of targeting moieties to plasmids using a convenient strategy. After sequence-specific winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, the extremities of a triplex-forming oligonucleotide hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a hairpin oligonucleotide using T4 DNA ligase. Any targeting moiety may be attached to the hairpin oligonucleotide. This strategy was used to attach an NLS peptide to a luciferase-expressing plasmid. Despite the presence of the padlock oligonucleotide, the reporter gene was efficiently expressed after transfection of the plasmid in HeLa or T24 cells, using either cationic lipids or cationic polymers as transfecting agents. However, no increase in gene expression could be observed as a result of peptide attachment. Nevertheless, the coupling strategy described in this paper may find applications as a tool for plasmid functionalization in other targeting experiments, and may lead to the development of improved vectors for gene therapy.

Amino Acid Sequence↗
PubMed1987

A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells.

Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed2003

Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures.

The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l(-1) were shown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.

Amino Acid Sequence↗
PubMed2006

Recombinant bovine herpesvirus 4 (BoHV-4) expressing glycoprotein D of BoHV-1 is immunogenic and elicits serum-neutralizing antibodies against BoHV-1 in a rabbit model.

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Starting from BoHV-4 cloned as a bacterial artificial chromosome (BAC), we used MuA transposase-mediated in vitro transposition to generate recombinant BoHV-4 expressing the immunodominant glycoprotein D (gD) of BoHV-1, one of the most important pathogens of cattle. Although a cis-acting element from woodchuck hepatitis virus (the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) in the 3' end of the gD expression cassette was required for maximal gD expression from plasmids in transient transfection assays, this element was not necessary for efficient expression of gD from recombinant BoHV-4 genomes. BoHV-4 recombinants containing gD expression cassettes with or without the WPRE expressed gD at similarly high levels. Several cell lines originating from different animal species expressed gD when infected with BoHV-4 recombinants. When rabbits were immunized with one of the recombinants, high levels of serum neutralizing antibodies against BoHV-1 were generated. This work is one of the first demonstrations of the use BoHV-4 as a vector for vaccine purposes and may provide the basis for BoHV-1 vaccination of cattle with recombinant BoHV-4.

Animals↗
PubMed2003

Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines.

We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

Animals↗
PubMed1970

Energy generation and utilization in hydrogen bacteria.

Studies on the relationship between cell synthesis and energy utilization in Hydrogenomonas eutropha have shown that the amount of oxidative energy required for synthetic reactions depends on the conditions of growth. The energy of hydrogen oxidation was most efficiently used when growth conditions were optimal (continuous culture, cells in exponential growth phase) and when the rate of growth was limited by H(2) or O(2) supply. Under these conditions, 2 to 2.5 atoms of oxygen were consumed by the oxyhydrogen reaction for the concomitant conversion of 1 mole of CO(2) to cell matter. This conversion efficiency, expressed as the O/C energyyield value, was observed with continuous cultures. A less efficient conversion was found with batch cultures. With limiting concentrations of CO(2) the rate of hydrogen oxidation was relatively high, and the O/C value was dependent on the growth rate. With nonlimiting concentrations of CO(2), the rate of hydrogen oxidation was strictly proportional to the rate of CO(2) fixation, and the O/C value was independent of growth rate. This proportionality between the rate of H(2) oxidation and the rate of CO(2) fixation suggested that energy supply regulates the (maximum) rate of growth. From the energy-yield measurements, we concluded that the oxidation of 1 mole of H(2) yields the equivalent of 2 moles of adenosine triphosphate for H. eutropha, and that at least 5 moles of this high-energy phosphate is required for the conversion of 1 mole of CO(2) into cellular constituents.

Carbon Dioxide↗
PubMed1989

Mutagenesis of the rat insulin II 5'-flanking region defines sequences important for expression in HIT cells.

To define the cis-acting elements important for rat insulin II gene expression, we analyzed the effects of 5' deletions and linker-scanning mutations on the expression of a rat insulin II reporter gene in an insulinoma cell line (HIT). The reporter gene contained 448 base pairs of 5'-flanking sequence joined to the bacterial chloramphenicol acetyltransferase gene. Expression of the 5' deletion mutations indicated that the minimal sequence requirement for efficient expression was 218 base pairs of 5'-flanking sequence, and at least three regions downstream from - 218 were important for transcription. A more precise localization of these elements and the cis-acting sequences in the promoter was achieved by analysis of the expression of 18 linker-scanning mutations. In these studies at least four other regions important for expression of the rat insulin II gene were identified. These findings suggest that the sequences important for rat insulin II and rat insulin I expression may differ significantly despite the high degree of sequence similarity in their 5'-flanking regions.

Animals↗