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A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinson's disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) (approximately 2 ng GDNF/h/10(5) cells). MPTP was administered (1 mg/kg/day, SC) for 7-10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the "Parkinson's score") had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [11C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [11C]raclopride to the dopamine D2-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

Purpose: NC100150 is a new type of ultra-small iron oxide (USPIO) blood pool agent. The aim of this study was to compare NC100150-enhanced MR angiography (MRA) to time-of-flight (TOF) MRA, phase-contrast (PC) MRA, and digital subtraction angiography (DSA) in experimental stenoses in pigs, and furthermore to determine the optimal dose of the contrast agent., Material and Methods: An experimental stenosis of the right and left common carotid artery (CCA) was applied surgically in 6 Yorkshire pigs. DSA was performed as the gold standard, followed by 3D-TOF, 3D-PC, and NC100150-enhanced MRA., Results: Eleven stenoses of the CCA were successfully applied. The degree of the stenosis, determined by NC100150-enhanced MRA, did not differ significantly from DSA, whereas TOF and PC MRA underestimated it. The image quality of the NC100150-enhanced MRA was superior to both TOF and PC MRA. The optimal dose of NC100150 was 5-6 mg Fe/kg, since higher doses did not further increase signal-to-noise ratio or contrast-to-noise ratio. There was a linear relationship between blood relaxation rate and contrast dose., Conclusion: NC100150-enhanced MRA has the potential for quantification of carotid stenoses and provides an alternative to DSA. The optimal dose of NC100150 was 5-6 mg Fe/kg.

The authors measured cerebral blood flow (CBF) and cerebral blood volume (CBV) by bolus tracking of a novel ultrasmall superparamagnetic iron oxide (USPIO) contrast agent (NC100150) and compared absolute and relative perfusion measurements with those obtained by a standard gadolinium-based contrast agent. They found a linear correlation between the two methods. A dose of 0.4 mg Fe/kg body weight was found to produce a signal drop similar to that of a standard 0.2 mmol/kg gadodiamide injection using spin-echo echoplanar imaging (SE-EPI) at 1.0 T. The measurements showed a high degree of reproducibility of repeated absolute as well as relative CBF and CBV values, lending further hope to the possibility of using magnetic resonance bolus tracking for routine CBF and CBV measurements. Finally, the authors present their initial experience with high-resolution, non-EPI CBV maps obtained from steady-state levels of an intravascular superparamagnetic contrast agent.

During an 11 month period, use of coercion was prospectively registered in a psychiatric department. Of all patients who had been subjected to coercion 36% were (n = 86) interviewed subsequently. Eleven percent of the interviewed patients did not know, that they had been submitted to coercion, 22% did not know the reason and 30% did not agree with the motivation for the decision. Forty-seven percent were satisfied with the information they had been given concerning how to complain of their treatment, 50% were satisfied with their adviser and 70% were satisfied with their overall admission. Sixty-five percent accepted that there should be a law allowing the use of coercion in psychiatry.

The study describes the type and amount of coercion used during 11 months of 1992. Four hundred and fourty-one cases were included. Young patients i.e. 10 to 29 years old, demented patients and patients suffering from organic psychoses were, overall, more often subjected to coercive measures. When looking at each type of coercion separately, it was found that detainment, compulsive treatment and fixation were especially used on the young and on patients suffering from schizophrenia. However, fixation used as a protective measure was mainly used on the demented and the 60 to 79 year-old patients.

We have studied the effect of a nonsedating antihistamine, cetirizine dihydrochloride, on the in vitro chemotaxis of leukocytes from human peripheral blood. We observed that 0.25 microgram/ml of cetirizine dihydrochloride in vitro significantly inhibited the chemotaxis of monocytes toward N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4. Higher concentrations of cetirizine, 1.0 and 2.5 micrograms/ml, completely inhibited monocyte chemotaxis without affecting cell viability. T-lymphocyte migration was also significantly depressed but not abolished. Pyrilamine (mepyramine) was not inhibitory in equimolar concentrations. According to these in vitro observations, we extended our studies to measure monocyte and T-lymphocyte chemotaxis in an open study, where four healthy volunteers and six patients with atopic dermatitis took 10 and 20 mg/day cetirizine 3 days. We observed a reduction in ex vivo monocyte and T-lymphocyte chemotaxis toward N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 without a reduction of the blood cell count. The results were confirmed in an ensuing double-blind, placebo-controlled study of 16 healthy subjects and 14 adult patients with atopic dermatitis, where ex vivo monocyte chemotaxis was reduced or abolished during cetirizine therapy. Serum levels of the two eosinophil-derived granule proteins, eosinophilcationic protein P and eosinophil protein X, were not changed during the treatment period of 7 days. The results show that cetirizine dihydrochloride has an inhibitory effect on monocytes and T lymphocytes in vitro and ex vivo. Our findings support the clinical observations that cetirizine dihydrochloride has an antiinflammatory effect besides its H1-blocking activity.

Security, say the authors, should be viewed as a profit center that protects the patients, staff, visitors, property, and assets of healthcare organizations from violent crime, injury, theft, or vandalism ... and the resulting devastating litigation. Security services, which protect the "bottom line," should be a central part of any healthcare organization.

The emergence of a wide variety of biological expression systems for the large-scale production of therapeutic proteins has shifted the focus from vectors to host organisms. Although expression systems now span bacteria, fungi, plants, insects, and mammalian cells, the vast majority of recombinant-derived biopharmaceuticals at the present time have been produced in Escherichia coli and in mammalian cells. This promises to change as the economic benefits of the newer systems permit the development of a new generation of proteins heretofore considered unfeasible for commercial development. Despite the impressive results which have been observed for many of the newer systems, there are many commercial considerations which suggest that E. coli and CHO cell expression systems may continue to dominate the manufacture of biopharmaceuticals for a long time to come.

This article presents the results of two investigations, each measuring cellular immune function on 3 investigation days 1 week apart in 15 high and 15 low hypnotizable healthy subjects randomly selected for three groups: (1) a guided imagery group receiving instructions to enhance cellular immune function: (2) a relaxation group which did not receive instructions regarding the immune system, and (3) a control group. Study 1 investigated changes in monocyte chemotaxis (MC) and lymphocyte proliferative response (LPR) to three mitogens, while natural killer cell activity (NKCA) was measured in study 2. The results show similar patterns of brief decreases in LPR and NKCA immediately after intervention on all investigation days in both the imagery and relaxation groups. Increases in MC were found in both intervention groups on day 1. On a follow-up investigation day in study 2, a brief stress task yielded a slight increase in NKCA. In study 2, the control group showed decreases in NKCA similar to those observed in the two intervention groups. In general there were no significant changes in preintervention immune function throughout the investigation period. When comparing the effects in high and low hypnotizable subjects, we found that high hypnotizables showed greater decreases in LPR and NKCA than low hypnotizables. There are several inconsistencies between the results of the limited number of investigations studying the effects of guided imagery and relaxation on immune function. These differences may in part be explained by differences in methodology, time intervals between blood sampling, and subject characteristics such as age, health status and hypnotizability. The inconsistent results make it premature to make inferences about possible benefits of the application of these techniques in the treatment of immune related diseases, and further investigations are needed.

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight. Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 and vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Dihydrofolate reductase (DHFR) cDNA sequences were isolated from a methotrexate-resistant mouse L5178Y cell line previously shown to contain methotrexate-resistant dihydrofolate reductase enzyme activity. Specifically-primed reverse transcription products were amplified using the polymerase chain reaction and then cloned into a mammalian expression plasmid. Candidate clones were identified by restriction analysis and then functionally tested by transfection into mouse 3T3 fibroblasts, selecting for methotrexate-resistant colonies. Sequence analysis of the cDNA clones demonstrated the substitution of tryptophan (TGG) in place of the wild-type phenylalanine (TTC) at codon 31. Sequencing of PCR-amplified genomic DNA extracted from the drug-resistant L5178Y cells confirmed the tryptophan codon at position 31. Transfection of mammalian tissue culture cells with expression plasmids containing the trp31 DHFR sequence resulted in substantial methotrexate-resistant colony formation. Recombinant trp31 DHFR enzyme activity expressed in stably-transfected Chinese hamster ovary cells was approximately 20-fold less sensitive to methotrexate inhibition than wild-type mouse DHFR enzyme activity. We conclude that the cloned Trp31 DHFR sequence encodes an enzyme substantially resistant to methotrexate which confers a drug-resistance phenotype to cells in which it is expressed.

We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.

Initiation of translation of eukaryotic mRNAs typically occurs at the first AUG triplet from the 5' end of the message, although several notable exceptions have been described. Using vectors which efficiently express the gene encoding the surface antigen of hepatitis B virus in monkey cells, we have studied the consequences of inserting ATG triplets in all three reading frames upstream of the usual translational initiation codon of this gene. In agreement with the scanning model for eukaryotic translation initiation, these additional codons can severely depress the initiation of translation at the 'authentic' start codon, although the extent of inhibition depends on sequences flanking the upstream AUG. Such inhibition can, however, be at least partially suppressed by the presence of a translation termination codon in-frame with the upstream AUG. These results raise the possibility that mammalian ribosomes can reinitiate translation at an AUG codon after previously initiating, and terminating, at an upstream site.

A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2-terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2-terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic.

We have isolated and determined the nucleotide sequence of a 1,626-base-pair fragment from R-plasmid R483 which encodes a trimethoprim-resistant dihydrofolate reductase. Analysis of the nucleotide sequence of this fragment revealed the presence of two open reading frames, each sufficient to encode polypeptides of approximately 17,000 daltons. Both open regions are preceded by sequences conforming closely to the canonical description of procaryotic promoters. A 490-base-pair HpaI fragment spanning one of the potential coding regions was inserted into a plasmid vector under the transcriptional control of the trp promoter. Cells transformed with this plasmid were trimethoprim resistant and produced dihydrofolate reductase activity which in vitro was resistant to moderate levels of trimethoprim. Analysis of the predicted amino acid sequence of this protein indicated that the R483-encoded trimethoprim-resistant enzyme was distantly related to the trimethoprim-sensitive bacterial homologs. The conserved amino acids were localized primarily to the region of the enzyme previously shown to comprise the hydrophobic substrate binding pocket.

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.

DNA clones encoding the complete 2,351 amino acid sequence for human factor VIII have been isolated and used to produce biologically active factor VIII in cultured mammalian cells. The recombinant protein corrects the clotting time of plasma from haemophiliacs and has many of the biochemical and immunological characteristics of serum-derived factor VIII.

The gene for glycoprotein D of herpes simplex virus type 1 (HSV-1) was expressed in stable mammalian cell lines. Glycoprotein D produced in these cells has a number of antigenic determinants in common with the native glycoprotein. Cell lines expressing glycoprotein D were used in an enzyme-linked immunosorbent assay to detect human antibodies to glycoprotein D. This strategy should prove useful in determining the extent to which the immune response to HSV-1 is directed toward glycoprotein D.

We have determined the sequence of the 1000 base pairs of DNA preceding the coding region of the mouse dihydrofolate reductase gene. 700 base pairs upstream of the translation start codon (position + 1) is the sequence CAACT, separated by 50 base pairs from the sequence TAATAA; these sequences resemble controlling elements responsible for accurate and efficient transcription initiation in a variety of eukaryotic genes. The region between -244 and -101 consists of a 3-fold tandem repeat of a 48-base pair sequence. S1 mapping results indicate that the 5' termini of the multiple dihydrofolate reductase mRNAs are heterogeneous, with the major terminus at -115 and with minor termini in the regions of -275 and -450. In addition, a portion of the 5'-ward region of the gene from -543 to -405 is represented differentially in some, but not all, mouse dihydrofolate reductase mRNAs. The above findings are consistent with the presence of two transcription promoter elements in the 5' end of the mouse dihydrofolate reductase gene. This has been substantiated by inserting portions of the 5'-ward 1000 base pairs of the gene into modular dihydrofolate reductase plasmids. When such constructs are transfected into Chinese hamster ovary cells lacking dihydrofolate reductase, function can be restored with equal transfection frequency when sequences surrounding the 5' CAACT-TAATAA region at -700 or a complete 48-base pair repeat from the region of -148 to -101 are present in the plasmid construct.

Results of a comprehensive dental examination, including a histologic evaluation, of a patient showing unilateral odontodysplasia revealed no hint as to the origin of the abnormality. A complete medical examination also resulted in negative findings, with the excetion of congenital bilateral talipes equinovarus. The genetic history was negative, and results of chromosomal studies of the child and her mother revealed no abnormalities. Neither had an anatomic or a functional abnormality, with the exception of the odontodysplasia in the child. Results of laboratory studies including complete blood counts, urinalyses, ferric chloride tests of the urine, and electrolyte tests also were negative. The special considerations given in this case in an attempt to establish a possible causation of the abnormality were inconclusive. We think, however, that the management of the patient was successful in regard to the prosthetic appliance, and every attempt will be made to follow up the patient to ensure a good prognosis of the remaining developing dentition.

A cDNA sequence which contains the entire coding region for human purine nucleoside phosphorylase (PNP) was recombined for selection and expression in mammalian cells. Plasmids containing either the simian virus 40 early promoter or the mouse metallothionein promoter positioned just upstream of the PNP coding sequence were constructed. These plasmids also contained the gene for a methotrexate-resistant dihydrofolate reductase, allowing for selection and amplification of positive transferrents after transfection of cells by the DNA-calcium phosphate coprecipitation technique. Expression of human PNP activity was readily detected in both mouse (L) and CHO cells by isoelectric focusing of cell extracts followed by histochemical staining for PNP activity. The simian virus 40 early promoter directed considerable expression of human PNP activity in CHO cells but only scant activity in mouse cells. The mouse metallothionein promoter was not successful in effecting human PNP expression in CHO cells but provided substantial human PNP activity in mouse cells and was inducible by incubation with zinc. HeLa cell transferrents were isolated and screened for the presence of transferred PNP cDNA sequences by Southern hybridization analysis. RNA transcripts derived from the transferred PNP cDNA were identified in one of these cell lines.

Pulse-labeling of vesicular stomatitis virus-infected HeLa and BHK cells with [3H]uridine throughout the infectious cycle demonstrated two peaks of uridine incorporation into virus-specific RNA molecules. By separating total RNA synthesis into replication and transcription products, we showed that replication occurs over a shorter period of time in one peak synthesis. The biphasic nature of uridine incorporation is in part due to a general membrane phenomenon of reduced metabolite transport during vesicular stomatis virus infection and in part due to the apparent uncoupling of replication and transcription. A change in the ratio of newly synthesized plus and minus strands of the genome length (42S) RNA was found as the infection proceeded. Early in the infection, plus-stranded 42S RNA comprised 40% of the total genome length RNA synthesis, whereas late in infection, only 15 to 20% of the 42S RNA synthesized was complementary to the virion minus strand. Our data suggest that the rate of synthesis of plus-stranded 42S RNA was constant throughout the infection. The rate of virus release was determined by monitoring the uptake of [3H]uridine into released virus particles. Virus maturation and release are closely associated with the assembly of 42S RNA-containing nucleocapsids.

We examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COS cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). We constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

Hepatitis B surface antigen (HBsAg) particles are secreted by Chinese hamster ovary cells that are stably transfected with the S gene of hepatitis B virus. The assembly of HBsAg into cylindrical and spherical particles occurred intracellularly within the endoplasmic reticulum. HBsAg particles accumulated within large dilated areas of the endoplasmic reticulum and remained within these structures for most of the time prior to secretion from the cells. Once the particles were formed, the HBsAg polypeptides did not appear to become associated with subsequent intracellular organelle membranes or the plasma membrane. HBsAg within the dilated structures did not bind wheat germ agglutinin, indicating that its oligosaccharide chains had not yet been processed to the complex form (containing terminal sialic acid-N-acetylglucosamine residues). The oligosaccharide chains of HBsAg are processed to the complex form and can be detected on the HBsAg after secretion, but this event was not detected within cells. In addition, HBsAg was not observed on the cell surface by indirect immunofluorescence or immunoprecipitation, although immunoelectron microscopy revealed some staining at or near the cell surface. These results suggested that HBsAg was either secreted from cells without being incorporated into the plasma membrane, or that the levels of HBsAg in the plasma membrane were below the limits of detection.

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexes were isolated in nonequilibrium Renografin density gradients. These nascent RNPs had the same buoyant density as virion nucleocapsids in both isopycnic Renografin and CsCl gradients. Both transcribing and replicating RNP complexes were shown to be stable in sucrose gradients, whereas only replicating RNP complexes were stable in Renografin gradients. Size analysis of the 5-min-pulse-labeled RNA species from the replicating RNPs using methylmercury gels revealed that the nascent strands were primarily less than full-length molecules. Longer times of radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA, which was primarily of the same sense as the virion strand when it was radiolabeled at 5 h postinfection. The percentage of this radiolabeled RNA which was plus stranded was higher at 2.5 h postinfection, reflective of the shift in plus- to minus-stranded full-length 42S RNA synthesis which occurs in the cell. Addition of cycloheximide to the infected cells before the addition of the radiolabel prevented the formation of these RNP complexes. Both the change in the percentage of minus strands found in the RNP complexes at the different times postinfection and the sensitivity to cycloheximide indicate that the RNP complex which was isolated was indeed the replicative complex.

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.

We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.