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Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

Although RAG-1 and RAG-2 have been shown to be indispensible for V(D)J recombination, their exact role in this reaction remains unclear. Co-transfecting RAG-1 and RAG-2 expression vectors into NIH3T3 fibroblasts confers V(D)J recombination activity to these otherwise recombinationally inactive cells. In this report we have found that in transient transfections of mouse NIH3T3 fibroblasts with RAG-1 and RAG-2 and the appropriate recombination substrates, one RAG-1 expression vector, pRAG-1A, is capable of yielding both signal joints and coding joints, while another RAG-1 expression vector, pRAG-1B, yields only signal joints. The RAG-1 open reading frame for these two expression vectors is interchangeable, indicating that the inability to resolve coding joints is due to the 45-base pair difference found in the 5'-untranslated regions of these constructs. Differences in this region result in a 15-fold difference in gene expression when the luciferase coding region is substituted for the RAG-1 cDNA. This report provides evidence that RAG-1 may have a role in both the initiation of V(D)J recombination as well as the resolution of coding ends. The data also suggest that these RAG-1 activities may be dependent on different levels of RAG-1 expression.

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenvironment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse lambda light chain genes which arose in response to a specific antigen. Laboratory mice possess three V lambda subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single lambda light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V lambda 1 genes from these single cells for mutational analysis. None of the 32 V lambda 1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other V lambda subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.

Site-specific recombination reactions in higher eukaryotes are uncommon, perhaps because of the potential genomic instability that they may create. We focus this review on the issues of site-specificity, reaction fidelity and immunologic diversity in the V(D)J recombination reaction.

Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair. Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures. Here, we describe the cloning of the murine FEN-1 gene. The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2. We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases. The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair. The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.

We used the polymerase chain reaction (PCR) to amplify genes encoding murine immunoglobulin (Ig) lambda light-chain variable (V) regions, using DNA isolated from populations of germinal center B-cells, to study somatic hypermutation at this locus. Sequence analysis revealed that 30% of the amplified products were chimeric molecules consisting of segments of the V lambda 1 and V lambda 2 genes. Furthermore, an amplification- and cloning-associated artifact exchanged sequences between mutational variants of V lambda 1 genes. These PCR artifacts interfere with the analysis of somatic hypermutation of Ig genes. An alternative method that avoids these artifacts is suggested which involves the amplification of individual V lambda genes from single cells.

Transcription, DNA topoisomerases and genetic recombination are interrelated for several structural reasons. Transcription can affect DNA topology, resulting in effects on recombination. It can also affect the chromatin structure in which the DNA resides. Topoisomerases can affect DNA and/or chromatin structure influencing the recombination potential at a given site. Here we briefly review the extent to which homologous direct repeat recombination and site-specific recombination in eukaryotes are affected by transcription and topoisomerases. In some cases, transcription or the absence of topoisomerases have little or no effect on recombination. In others, they are important components in the recombinational process. The common denominator of any effects of transcription and topoisomerases on recombination is their shared role in altering DNA topology.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.

Ataxia telangiectasia (AT) and Bloom's syndrome (BS) patients are characterized by sensitivity to radiation, increased lymphoid malignancy, and frequent translocations to the antigen receptor loci. Because of these features, there has been a persistent question as to whether the V(D)J recombinase might be abnormal in cells from these patients. Such abnormalities might be due to inappropriate to inaccurate expression of components of the V(D)J recombinase or due to mutation in a component shared between V(D)J recombination and other cellular processes, such as DNA repair. Bloom's syndrome is associated with a ligation deficiency, and this activity may contribute in the end resolution steps of both site-specific and general DNA-processing reactions. In the current study, we have activated V(D)J recombination in normal, AT, and BS fibroblasts and in fibroblasts from a patient with mutations that largely abolish DNA ligase I activity. We find that the signal and coding joint formation of the V(D)J recombination reaction are entirely normal in AT, BS, and DNA ligase I mutant cells. In addition to ruling out abnormalities of the V(D)J recombinase in AT, BS, and DNA ligase I mutant cells, these studies suggest that DNA ligase I is unlikely to be required for signal or coding end joining in the V(D)J recombination reaction.

In V(D)J recombination, two site-specific cuts are made adjacent to V, D, and J subexons to create four DNA ends, two of which (the coding ends) are joined to generate the exon that encodes the variable domain of the antigen receptor. Although deviations from consensus signal sequences have been reported previously to have a large impact on the efficiency of V(D)J recombination, coding end sequence has been assumed to be neutral with respect to the efficiency of recombination. We have used extrachromosomal V(D)J recombination substrates to undertake a systematic comparison of coding end sequences. Substrates were constructed that contain identical consensus recombination signal sequences, where only the coding ends were varied. Surprisingly, we found that nucleotide sequence at the coding end can affect the efficiency of V(D)J recombination > 250-fold. Variable initiation of recombination appears to account for most of the effect. This finding has mechanistic implications because it indicates that signal-binding proteins involved in V(D)J recombination may have different levels of activity when confronted with coding ends of different sequence. Our results also indicate that coding end sequence must be considered to be among the major factors that shape the antigen receptor repertoire.

Substrates for studying V(D)J recombination in human cells and two human pre-B-cell lines that have active V(D)J recombination activity are described. Using these substrates, we have been able to analyze the relative efficiency of signal joint and coding joint formation. Coding joint formation was five- to sixfold less efficient than signal joint formation in both cell lines. This imbalance between the two halves of the reaction was demonstrated on deletional substrates, where each joint is assayed individually. In both cell lines, the inversional reaction (which requires formation of both a signal and a coding joint) was more than 20-fold less efficient than signal joint formation alone. The signal and coding sequences are identical in all of these substrates. Hence, the basis for these differential reaction ratios appears to be that coding joint and signal joint formation are both inefficient and their combined effects are such that inversions (two-joint reactions) reflect the product of these inefficiencies. Physiologically, these results have two implications. First, they show how signal and coding joint formation efficiencies can affect the ratio of deletional to inversional products at endogenous loci. Second, the fact that not all signal and coding joints go to completion implies that the recombinase is generating numerous broken ends. Such unresolved ends may participate in pathologic chromosomal rearrangements even when the other half of the same reaction may have proceeded to resolution.

Among site-directed DNA recombination systems, V(D)J recombination is noteworthy in that identical reactants yield different recombination products at the junction of joined segments. This variation is the basis for diversity at the base of antigen receptor binding pockets and corresponds to V-(D)-J DNA junctions. An abundance of certain junctions has been noted. It has been proposed that these junctions are favoured because they occur where short regions of homology in participating coding ends might align preferentially. Here we use a system that is entirely free from cellular selection to show that the diversity of coding joints can be severely restricted when the coding ends participating in the reaction have short regions of homology. This constraint on diversity is diminished but not eliminated by terminal deoxynucleotidyl transferase, a mechanistic feature that has implications for the establishment of the immune repertoire.

V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.

The V(D)J recombination reaction is directed by a pair of signal sequences, each consisting of a palindromic heptamer, an A/T-rich nonamer, and an intervening spacer sequence of 12 or 23 base pairs. The spacer sequence previously has not been analyzed for a functional role. In this study, numerous simultaneous sequence changes have been made in the spacer of each signal to test their functional importance. All of the AT base pairs in each signal were changed to GC base pairs. This particular change is of interest because it markedly increases the energy that would be required to melt out the two strands of each signal to permit the intersignal base pairing proposed in a commonly invoked model for signal-signal interaction in V(D)J recombination. We find that changing 6 to 12 AT base pairs in the 12-signal to GC does not affect V(D)J recombination, nor does changing 11 of 23 AT base pairs in the 23-signal. Substrates with all-GC spacer sequences in both the 12- and the 23-signal also recombine at efficiencies that are not significantly reduced. These studies demonstrate that the sequences at these particular positions are not recognized by the recombinase. In addition, the data do not support models invoking signal-signal base pairing.

Murine severe combined immune deficiency (scid) is marked by a 5,000-fold reduction in coding joint formation in V(D)J recombination of antigen receptors. Others have demonstrated a sensitivity to double-strand breaks generated by ionizing radiation and bleomycin. We were interested in establishing the extent of the defect in intramolecular and intermolecular DNA end joining in lymphoid and nonlymphoid cells from scid mice. We conducted a series of studies probing the ability of these cells to resolve free ends of linear DNA molecules having various biochemical end configurations. We find that the stable integration of linear DNA into scid fibroblasts is reduced 11- to 75-fold compared with that in normal fibroblasts. In contrast, intramolecular and intermolecular end joining occur at normal frequencies in scid lymphocytes and fibroblasts. This normal level of end joining is observed regardless of the type of overhang and regardless of the requirement for nucleolytic activities prior to ligation. The fact that free ends having a wide variety of end configurations are recircularized normally in scid cells rules out certain models for the defect in scid. We discuss the types of DNA end joining reactions that are and are not affected in this double-strand break repair defect in the context of a hairpin model for V(D)J recombination.

It has been shown previously by others that transcription is temporally correlated with the onset of V(D)J recombination at the endogenous antigen receptor loci. We have been interested in determining whether this temporal correlation indicates a causal connection between these two processes. We have compared V(D)J recombination minichromosome substrates that have transcripts running through the recombination zone with substrates that do not in a transient transfection assay. In this system, the substrates acquire a minichromosome conformation within the first several hours after transfection. We find that the substrates recombine equally well over a 100-fold range in transcriptional variation. In additional studies, we have taken substrates that have low levels of transcription and inhibited transcription further by methylating the substrate DNA or by treating the cells with a general transcription inhibitor (alpha-amanitin). Although these treatments decrease the level of expression an additional 10-100-fold, there is still no observable effect on V(D)J recombination. Based on these results, we conclude that transcription is not necessary for the V(D)J reaction mechanism and does not alter substrate structure at the DNA level or at the simplest levels of chromatin structure in a way that affects the reaction.

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.

The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100-fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromosome DNA. The minichromosomes acquire resistance to both the intracellular V(D)J recombinase and exogenous endonuclease only after DNA replication. Therefore, CpG methylation specifies a chromatin structure that, upon DNA replication, is resistant to eukaryotic site-specific recombination. These findings are important to V(D)J recombination as well as to the chromatin assembly of methylated DNA during replication.

Site-specific DNA recombination has been identified in a wide variety of biological systems. In vertebrates, however, the only identified use of this genetic device is in the immune system. Here it plays a critical role in generating a diverse repertoire of surface receptors to intercept invading microbes and parasites. The mechanism and orchestration of this reaction are intriguing and are relevant to a broad array of related biological and biomedical issues.

We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.

A high-pressure liquid chromatographic (HPLC) method using a micro-particulate silica column and gradient elution was developed that separated 12-O-tetradecanoylphorbol-13-acetate (TPA) from 20-oxo-TPA; 12-O-tetradecanoylphorbol (TP); 13-O-acetylphorbol (PA), and from the diterpene alcohol, phorbol (P). A series of other phorbol-ester tumor promoters were also separated via HPLC. Spectrophotometric determination at 232 nm allowed detection sensitivities of 0.05 microgram of TPA. When tritiated TPA was applied to mouse skin, the majority of the tritiated product recovered was TPA, indicating only minimal metabolism of TPA and no need for metabolic activation for tumor promotion.

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either never greater than 0.5 nm in radius, much smaller in number if they were greater than 0.5 nm in radius, or shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.

Solid and papillary epithelial neoplasms of the pancreas from six female patients were studied using immunohistochemistry and electron microscopy to define better their histogenesis. The tumors ranged in diameter from 5 to 15 cm (average: 9 cm), and, on cross section, most had areas of hemorrhage and necrosis, sometimes extensive. Microscopically, there was a solid and pseudopapillary pattern, with tumor cells typically having ovoid nuclei with delicate folding and indistinct nucleoli. Of note were the following: a relatively low mitotic rate (range: 0-6/20 hpf), the presence of hyaline globules (four of six cases), and collections of foam cells (three of six cases). Staining for cytoplasmic argyrophil granules was negative in each case. Ultrastructurally, the solid and papillary epithelial neoplasms of the pancreas showed evidence of acinar or ductular differentiation. Two contained zymogen granules, one had intermediate filaments (probably keratin), and three had abundant rough endoplasmic reticulum and mitochondria. Immunostaining was positive for chymotrypsin (six of six cases), trypsin (four of six), and amylase (three of six). None was positive for alpha-1-antitrypsin, neuron-specific enolase, pancreatic polypeptide, gastrin, glucagon, somatostatin, or insulin. The findings support an origin from exocrine pancreas, and follow-up indicates a low rate of malignancy, with local recurrence in two of the six patients.

The coding regions of antigen receptor genes assembled by variable-diversity-joining region [V(D)J] recombination are known in many cases to have undergone deletions of several nucleotides and also to contain insertions of noncoded nucleotides at the recombined junction (the coding joint). By using extrachromosomal recombination substrates to transfect lymphoid cell lines, we show that the signal joint (the fusion of the corresponding recognition signal sequences) can also contain insertions; however, nucleotide loss from the signals is very rare. The frequency of nucleotide addition varies among pre-B-cell lines in a manner proportional to their content of terminal deoxynucleotidyltransferase. We also find frequent nucleotide additions (and deletions) at coding joints, but in this case there is no strong correlation with the level of terminal deoxynucleotidyltransferase activity. Inserts at both signal and coding joints are rich in G + C, consistent with the base utilization preference of this enzyme.

Therapeutic gastric irradiation has been used to reduce peptic juice secretion in patients with peptic ulcer disease. Between 1937 and 1968 a total of 2049 patients received such therapy at the University of Chicago. Three of these patients are known to have developed sarcomas in the field of radiation. Two gastric leiomyosarcomas of the stomach were diagnosed 26 and 14 years after treatment and a malignant fibrous histiocytoma of the anterior chest wall was removed six years after gastric irradiation. Of 743 peptic ulcer patients treated without irradiation and constituted as a control group for the study of therapeutic gastric radiation, none is known to have developed sarcoma. As the incidence of sarcoma in these patient groups is known only from the tumor registry of the University of Chicago, other cases of sarcoma may exist in the groups. While an increased incidence of sarcoma has not been proven to occur in patients who received therapeutic gastric irradiation for peptic ulcer disease, the possibility of such a risk should be borne in mind by physicians caring for such patients.

The clinical, radiographic, and pathologic features of 10 patients with documented malignant small-cell tumor of the thoracopulmonary region (Askin tumor) were reviewed. The tumor represents a distinct pathologic entity of neuroectodermal origin. Clinically, it presents as a chest-wall mass with or without pain. Its radiographic appearance is that of a soft-tissue mass with or without pleural or rib involvement, often with metastatic disease--to the skeletal system, bone marrow, thorax, and sympathetic chain. Two patients developed metastases to the adrenal gland and liver, one after autologous bone marrow transplantation. The radiologist should be aware of this entity and its pattern of metastatic spread since metastases are treated aggressively.

We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.

The interaction of the amphipath chlorpromazine (CPZ) with the human erythrocyte membrane was evaluated. The partition coefficient of CPZ between the membrane bilayer and the aqueous compartment, measured spectrophotometrically, ranged between 1 and 3 X 10(3). An independent estimate, 4.6 X 10(3), was obtained by a novel method which avoided the measurement of binding and determined instead the variation of the hemolytic potency of the amphipath with the ratio of buffer volume to membrane volume. The maximal uptake of CPZ exceeded 2 X 10(9) molecules/red cell, corresponding to a volume greater than that of the bilayer itself. Such heavily loaded membranes were increased in thickness more than 2-fold, suggesting the formation of a CPZ-rich zone at the center of the bilayer. Ghosts loaded with massive levels of CPZ condensed approximately 20-fold in surface area and increased proportionately in thickness, suggesting the formation of a novel CPZ-lipid solution. CPZ caused hemolysis by a colloid-osmotic mechanism. By measuring the simultaneous uptake of mannitol and sucrose, we determined that CPZ induced holes of constant size but variable number. If circular, the holes would have had a diameter of approximately 14 A. The time-averaged number of holes ranged from 0.09 per cell (signifying intermittency) to 16. Freeze-fracture electron microscopy of CPZ-treated red cells revealed multiple round patches of nearly particle-free bilayer up to 0.3 micron in diameter with crowding of the intramembrane particles into the surrounding membrane. We interpret these images to signify lateral phase separation within the CPZ-treated bilayer. Hemolysis could, therefore, result from the intermittent opening of weak seams at phase boundaries; these could then be fluctuating slits approximately 14 A in width and of variable length, rather than simple circular holes.

We have examined the level of immunoglobulin gene V(D)J recombination activity in a number of cell lines derived from lymphoid or nonlymphoid lineages. The assay we employed uses extrachromosomal DNA as substrate and thereby avoids difficulties associated with the use of chromosomally integrated substrates. The recombination activity decreases during B-lymphoid development. It is highest at the earliest stages of committed B-cell differentiation and then falls progressively, reaching undetectable levels at the mature B-cell stage. The activity is also present in multipotential progenitors of myeloid cells and in pre-T cells but not mature T cells. No activity was found in several nonhematopoietic cell lines. Recombination was seen only among substrate molecules which had replicated in the eukaryotic cells. Several possible interpretations of this result are discussed.

We have undertaken a study of the mechanism of DNA transfer into primary chicken erythrocytes by a method named osmotic transfection. The cells are subjected to controlled osmotic swelling in NH4Cl and then ruptured in a lower osmotic strength solution containing DNA and DEAE-dextran. The osmotic rupture results in transient formation of a single hole in the cell membrane, which is followed within hours by recovery of near normal levels of RNA and protein synthesis. The association of DNA with the cells is much greater for ruptured than for unruptured cells or for cells that have been lysed and resealed before DNA is added. Transient formation of pores in the cell membrane is apparently essential for high rates of macromolecular transfer into the cell. DEAE-dextran increases the amount of DNA associated with the cells, especially after cell rupture. Our understanding of the mechanism has allowed us to extend the application of osmotic transfection to essentially all developmental stages of avian erythroid differentiation. Osmotic transfections were done with plasmids containing the chloramphenicol acetyl transferase (cat) gene placed between the chicken beta-globin promoter and the 3' beta-globin enhancer. The pattern of CAT expression at sequential developmental stages parallels that of the endogenous gene, showing that osmotically transfected cells appear to retain developmental fidelity. The approach provides a convenient, sensitive, and flexible system for the study of transient gene expression as a function of development.

Cyclosporine A (CSA)-dipalmitoylphosphatidylcholine (DPPC) interactions were investigated using scanning calorimetry, infrared spectroscopy, and Raman spectroscopy. CSA reduced both the temperature and the maximum heat capacity of the lipid bilayer gel-to-liquid crystalline phase transition; the relationship between the shift in transition temperature and CSA concentration indicates that the peptide does not partition ideally between DPPC gel and liquid crystalline phases. This nonideality can be accounted for by excluded volume interactions between peptide molecules. CSA exhibited a similar but much more pronounced effect on the pretransition; at concentrations of 1 mol % CSA the amplitude of the pretransition was less than 20% of its value in the pure lipid. Raman spectroscopy confirmed that the effects of CSA on the phase transitions are not accompanied by major structural alterations in either the lipid headgroup or acyl chain regions at temperatures away from the phase changes. Both infrared and Raman spectroscopic results demonstrated that CSA in the lipid bilayer exists largely in a beta-turn conformation, as expected from single crystal x-ray data; the lipid phase transition does not induce structural alterations in CSA. Although the polypeptide significantly affects DPPC model membrane bilayers, CSA neither inhibited hypotonic hemolysis nor caused erythrocyte hemolysis, in contrast to many chemical agents that are believed to act through membrane-mediated pathways. Thus, agents, such as CSA, that perturb phospholipid phase transitions do not necessarily cause functional changes in cell membranes.

Two conserved DNA sequences serve as joining signals in the assembly of immunoglobulins and T-cell receptors from V-, (D)-, and J-coding segments during lymphoid differentiation. We have examined V(D)J recombination as a function of joining signal sequence. Plasmid substrates with mutations in one or both of the heptamer-spacer-nonamer sequences were tested for recombination in a pre-B-cell line active in V(D)J recombination. No signal variant recombines more efficiently than the consensus forms of the joining signals. We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length greater than 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites.