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Three different techniques of instrumental analysis applied at ISAS were evaluated within a proficiency test, organised by IRMM in Geel. The 3 techniques, in particular flame atomic absorption spectrometry (FAAS), inductively coupled plasma mass spectrometry (ICP­MS) and total­reflection X­ray fluorescence (TXRF) were used for multielement trace analysis of a sediment sample after digestion. The reported results of up to 9 elements were evaluated with respect to the reference values after the disclosure of the latter. The relative deviations, mostly <4%, demonstrate the high accuracy of the 3 techniques. In addition, the uncertainty bars of report and reference show a high degree of overlapping, which gives evidence that the mean values are not significantly different. The high ranking of ISAS results among all 239 participants of the test confirms these findings. With regard to accuracy, ICP­MS and TXRF are somewhat inferior to conventional FAAS but in their favour they are labour­ and time­saving and have a good detection power.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the basis for the evolutionary conservation of myc gene family has remained unclear. To elucidate this issue, we have generated mice in which the endogenous c-myc coding sequences have been replaced with N-myc coding sequences. Strikingly, mice homozygous for this replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have evolved, to a large extent, to facilitate differential patterns of expression.

The intronic IgH enhancer E(mu), which consists of the core enhancer (cE(mu) flanked by 5' and 3' matrix attachment regions (MAR), has been implicated in the control of IgH locus recombination and transcription. Both cE(mu) and the MAR are required to enhance transcription of an IgH transgene. To elucidate the regulatory functions of cE(mu) versus its associated MAR in IgH class switch recombination (CSR), we have assayed ES cell lines which have targeted deletions of these elements, both individually and in combination, by the Rag-2-deficient blastocyst complementation method. Mutant B cells from chimeric mice were activated in culture and the influence of the mutations on CSR was assessed by analysis of B cell hybridomas. We find that the cE(mu) is necessary and sufficient for providing the functions of E(mu) required for efficient CSR at the IgH locus. However, the 5' and 3' MAR sequences, as well as the known I(mu) transcription start sites and the bulk of I(mu) coding sequences, were dispensable for the process.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre‐) B cell differentiation, we find that most Abelson murine leukemia virus (A‐MuLV)‐transformed pre‐B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A‐MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre‐B cell lines from normal mice, attempted V kappa‐to‐J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back‐to‐back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site‐specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

We have isolated a 12 kb clone from the murine genome which we show by DNA transfection studies to contain an entire functional L‐myc gene and the transcriptional promoter sequences necessary for its expression. We have also isolated a 3.1 kb cDNA sequence from a murine brain cDNA library which corresponds to most of the L‐myc mRNA. We have identified the L‐myc coding region within the genomic clone by a combination of S1 nuclease analyses. Northern blotting analyses and comparative nucleotide sequence analyses with the cDNA clone. The L‐myc gene appears to be organized similarly to the other well‐characterized myc‐family genes, c‐myc and N‐myc. The predicted amino acid coding sequence of the L‐myc gene indicates that the L‐myc protein is significantly smaller than c‐ and N‐myc, but is highly related. In particular, comparison of the N‐ and c‐myc protein sequences reveals seven relatively conserved regions interspersed among non‐conserved regions; the L‐myc gene retains five of these conserved regions but lacks two others. In addition, a portion of one highly conserved region is encoded within a different region of the L‐myc gene but, due to changes in the size of L‐myc exons relative to those of N‐ and c‐myc, maintains its overall position in the peptide backbone with respect to other conserved regions. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc‐family proteins.

During B cell development V kappa gene rearrangement seems to occur only in mu‐positive pre‐B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre‐B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre‐B cells.

Transgenic constructs containing the murine L‐myc gene under the control of the immunoglobulin transcriptional enhancer element (Emu) are expressed at unexpectedly high levels in thymocytes and proliferating T cells compared with cells from bone marrow and proliferating B cells. In contrast, double transgenic animals bearing constructs containing the L‐ and N‐myc genes similarly linked to the Emu element maintain preferential L‐myc expression in T cells but express the N‐myc transgene preferentially in B cells. These results indicate that the L‐myc gene contains elements that act in concert with the Emu element to allow preferential expression in T lineage cells. In correspondence to the expression pattern, Emu‐L‐myc transgenic mice show expanded thymic cortices and irregularly formed splenic follicles with expanded T cell areas. Moreover, the percentage of thymocytes positive for the surface marker 1C11, which defines thymic progenitor cells, activated T cells and preleukemic T cells, is dramatically raised in transgenic mice compared with normal littermates. Emu‐L‐myc transgenic animals are predisposed to clonal lymphoid tumors, most of which are T cell lymphomas. The relative incidence, latency period, and degree of malignancy of Emu‐L‐myc tumors compared with Emu‐N‐ or c‐myc tumors is consistent with a lower oncogenic potential of the L‐myc gene. However, the Emu‐L‐myc tumors do not express detectable levels of endogenous myc family genes indicating that the L‐myc protein can substitute for c‐ or N‐myc in the generation and growth of lymphoid neoplasms.

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N‐myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu‐N‐myc genes). High‐level expression of the E mu‐N‐myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu‐N‐myc lines reproducibly developed pre‐B‐ and B‐lymphoid malignancies. The E mu‐N‐myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu‐N‐myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu‐N‐myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N‐myc genes. In contrast, all of the E mu‐N‐myc cell lines had exceptionally low or undetectable levels of the c‐myc RNA sequences, consistent with the possibility that high‐level N‐myc expression can participate in the negative ‘cross‐regulation’ of c‐myc gene expression. Our findings demonstrate that deregulated expression of the N‐myc gene has potent oncogenic potential within the B‐lymphoid lineage despite the fact that the N‐myc gene has never been implicated in naturally occurring B‐lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

Prices of both nitrogen fertilizers and forages have increased in recent years causing concern about the profitability of fertilizing forage grasses. Yield response functions, which permit computation of economic optima, were estimated using regression analysis for Kentucky bluegrass (Poa pratensisL.), tall fescue (Festuca arundinaceaSchreb.) orchardgrass (Dactylis glomerataL.), reed canarygrass (Phalaris arundinaceaL.), smooth bromegrass (Bromus inermisLeyss.), and timothy (Phleum pratenseL.). Experimental data from 3 years, 1968, 1969, 1970, with N treatments of 0, 112, 224, and 448 kg/ha were used to estimate the regression equations. Economic analyses were made with 3 N prices, 44, 55, and 66 cents/kg, and 5 forage prices, 2.2, 3.3, 4.4, 5.5, and 6.6 cents/kg. The economically optimal N treatment varied by species and was sensitive to changes in N‐forage price ratio, declining as the N price rose or the forage price declined. Fertilization with N was found to be profitable for all but very unfavorable price ratios.

An extremly sensitive procedure for the determination of platinum in human body fluids is presented. A high pressure decomposition of the samples is followed by adsorptive voltammetric measurement. A detection limit down to 0.2 ng Pt/l sample allowed baseline levels of platinum in body fluids (urine: 0.5–15 ng/l, blood and blood plasma: =0.8–6.9 ng/l) to be evaluated. The concentration ranges in body fluids of occupationally exposed people were determined to 21–2900 ng/l (urine), 32–180 ng/l (blood) and 95–280 ng/l (blood plasma).

Transgenic constructs containing the murine L‐myc gene under the control of the immunoglobulin transcriptional enhancer element (Emu) are expressed at unexpectedly high levels in thymocytes and proliferating T cells compared with cells from bone marrow and proliferating B cells. In contrast, double transgenic animals bearing constructs containing the L‐ and N‐myc genes similarly linked to the Emu element maintain preferential L‐myc expression in T cells but express the N‐myc transgene preferentially in B cells. These results indicate that the L‐myc gene contains elements that act in concert with the Emu element to allow preferential expression in T lineage cells. In correspondence to the expression pattern, Emu‐L‐myc transgenic mice show expanded thymic cortices and irregularly formed splenic follicles with expanded T cell areas. Moreover, the percentage of thymocytes positive for the surface marker 1C11, which defines thymic progenitor cells, activated T cells and preleukemic T cells, is dramatically raised in transgenic mice compared with normal littermates. Emu‐L‐myc transgenic animals are predisposed to clonal lymphoid tumors, most of which are T cell lymphomas. The relative incidence, latency period, and degree of malignancy of Emu‐L‐myc tumors compared with Emu‐N‐ or c‐myc tumors is consistent with a lower oncogenic potential of the L‐myc gene. However, the Emu‐L‐myc tumors do not express detectable levels of endogenous myc family genes indicating that the L‐myc protein can substitute for c‐ or N‐myc in the generation and growth of lymphoid neoplasms.

The determination of Au, Pt, Pd and Rh at ng- and pg-levels in manganese crust, natural water and geological and biological standards is based upon extraction of these elements with Se via a coprecipitation technique, elaborated as a micro-technique. All steps of the combined procedure are controlled by radiotracer (195Au) and yields vary between 90 and 100%. Zeeman graphite furnace atomic absorption spectrometry and total reflection X-ray spectrometry are employed as determination steps. Sources of systematic errors are discussed.

We have isolated a cDNA copy of a truncated C gamma 2b transcript produced by Abelson murine leukemia virus transformants that spontaneously switch from mu to gamma 2b. The initiation site of this transcript was 2 kilobases 5' to the gamma 2b switch recombination region, demonstrating its germ line origin. Nucleotide sequence analyses suggest that this transcript does not encode a protein. Expression of germ line gamma 2b transcripts in Abelson murine leukemia virus transformants and in normal spleen cells correlated with endogenous gamma 2b class switch activity.

This research was undertaken to assess the influence of harvest schedules and fertilization on the persistence and productivity of four warm‐ and six cool‐season grass species under minimal temperature and moisture stresses. The grasses were clipped three, five, or eight times during each of 2 years. The first clipping each year was taken on April 26, May 7, or June 10 and aftermath clippings were taken at 21‐, 35‐, or 55‐day intervals, respectively. Ammonium nitrate was applied in three equal applications during each growing season. A total equivalent of 168 or 336 kg/ha was applied to each plot. Persistence of Kentucky bluegrass (Poa pratensisL.), tall fescue (Festuca arundinaceaSchreb.), orchardgrass (Dactylis glomerataL.), and Timothy (Phleum pratenseL.) improved as clipping frequency was increased from three to eight cuts per year, especially at the high rate of N. Reed canarygrass (Phalaris arundinaceaL.) and smooth bromegrass (Bromus inermisLeyss.) stands were better if clipping was less frequent. Stands of bermudagrass [Cynodon dactylon(L.) Pers.], indiangrass [Sorghastrum nutans(L.) Nash], big bluestem (Andropogon gerardiVitman) and switch‐grass (Panicum virgatumL.) had deteriorated badly after 2 years, regardless of treatment. Orchardgrass was the most productive species overall and was least affected by the clipping and fertilizer treatments. Dry matter yields of grasses in the second harvest year were differentially influenced by clipping and nitrogen fertilizer (3‐factor interaction = P < .005). At the high rate of N, orchardgrass, reed canarygrass, and smooth bromegrass yields were highest with three clippings, tall fescue and timothy yields were highest with five clippings, and Kentucky bluegrass yields were highest with eight clippings. At the low rate of N all species, except Kentucky bluegrass and timothy, produced highest yields with three clippings. Timothy yields were highest with five clippings, and clipping frequency had little effect on bluegrass yields. Yields were reduced most by infrequent clipping at the high rate of N (Kentucky bluegrass, tall fescue, timothy) and by frequent clipping at the low rate of N (all species except bluegrass). Persistence and yield distribution responses to clipping and nitrogen fertilizer were different from those commonly reported for lower elevations and were attributed to less temperature and moisture stress. Management requirements for warm‐season grasses differ from those for cool‐season grasses and need to be studied in more detail.

The reduction of the dioctylammonium ion pairs of the isomers of molybdatosilicic acid with Sn(II) was investigated by a photometric distribution titration in the system water/ chloroform. 2e- and 4e-reduction products were found. The 2e-ß-, 2e-a- and 4e-a-products are soluble in the organic phase, whereas the 4e-ß-product dissolves only in water.

We describe a system to evaluate the function of lymphocyte-specific and generally expressed genes in the differentiation and/or function of lymphocytes. RAG-2 (recombination-activating gene 2)-deficient mice have no mature B and T lymphocytes due to the inability to initiate VDJ recombination. Blastocysts from RAG-2-deficient mice generate animals with no mature B and T cells following implantation into foster mothers. However, injection of normal ES cells into RAG-2-deficient blastocysts leads to the generation of somatic chimeras with mature B and T cells all of which derive from the injected ES cells (referred to as RAG-2-deficient blastocyst complementation). Complementation of RAG-2-deficient blastocysts with mutant ES cells heterozygous for a targeted mutation that deletes all immunoglobulin heavy-chain joining (JH) gene segments (JH+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (JH-/-) generates animals with normal T cells but no B cells, due to a block in B-cell development at a very early stage. Transfection of a functionally assembled mu heavy-chain gene into the JH-/- ES cells prior to blastocyst injection rescues the JH-/- mutation and allows the generation of both mature T and mature B cells. The rescued B cells express IgM but not IgD and respond normally to bacterial lipopolysaccharide stimulation by proliferating and by secreting IgM.

Human neuroblastoma IMR-32 cells have large homogeneously staining regions (HSRs), primarily in the short arms of chromosome 1. We have constructed a recombinant phage library that is enriched for DNA present in the HSR of this chromosome by using fluorescence-activated flow sorting for initial chromosome purification. Eleven distinct cloned DNA segments were identified that showed significantly greater hybridization to IMR-32 genomic DNA, detected by Southern blotting, than to normal human genomic DNA. These sequences have also been localized to the HSR of chromosome 1 by in situ hybridization. Based on an approximate 50-fold sequence amplification for each cloned segment and a total HSR size of 150,000 kilobases, the amplified unit in the HSR is estimated to be 3,000 kilobases. Sequences homologous to all cloned HSR DNA segments were mapped to human chromosome 2 by using human-mouse hybrid cells. Further work using in situ hybridization demonstrated that cloned HSR segments were localized in the short arm of chromosome 2 in both normal and IMR-32 cells. Thus, the amplification of these sequences in IMR-32 cells may have involved transposition from chromosome 2 to chromosome I.

Immunoglobulin (Ig) gene expression has been followed during the later stages of development of the murine fetal liver. Biosynthetic labeling and immunoprecipitation were used to isolate Ig-related polypeptides from fetal and neonatal livers. By examination of the specific immune precipitates, the earliest detectable Ig was shown to consist only of mu heavy chain. At about the time of birth, when light chain synthesis became evident, separation of surface Ig-positive cells from surface Ig-negative cells by using anti-Ig-coated dishes showed that cells lacking surface Ig (pre-B lymphocytes) synthesized only mu chains. Thus, commencement of light chain synthesis was closely coordinated with the appearance of surface Ig. Ig RNA species were examined by electrophoretic fractionation and hybridization with cloned Ig DNA sequences. The sizes and amounts of Ig mRNA were found to correlate with the pattern of mu and light chain protein biosynthesis. mu chain RNA species appeared earlier in gestation than light chain RNA did, and only after birth did light chain sequences reach levels equivalent to those of mu chain. Cell populations enriched in pre-B lymphocytes also contained an excess of mu over light chain mRNA.

Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process.

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.

Germline C_H transcripts initiate from a promoter upstream of a non-coding I exon, proceed through the switch (S) region and terminate downstream of the associated C_H exons. To elucidate the role of germline transcription in Ig heavy chain class switch recombination (CSR), we used gene targeting in embryonic stem (ES) cells to replace most of the I_γ2b exon from immediately 3′ of the majority of transcription initiation sites to beyond its donor splice site with a PGK-neo^r gene inserted in the same transcriptional orientation as the endogenous unit. The mutation was introduced into both alleles of ES cell lines (referred to as I_γ2b^N/N) and the neo^r gene was deleted (referred to as I_γ2b^-/-) by the IoxP/Cre method. These mutations were assayed for effects on CSR in B cells derived via RAG-2-deficient blastocyst complementation. I_γ2b^-/- B cells lacked ability to switch to IgG2b both in vivo and in vitro, and, correspondingly, showed no germline transcription through the I_γ2b exon, S_γ2b region. In contrast, I_γ2b^N/N B cells switched at normal levels IgG2b and showed substantial transcription through the S_γ2b and C_γ2b regions. Taken together, these results show that the I_γ2b sequences, per se, are not necessary for mediating CSR since a transcribed PGK-neo^r gene can replace its function. However, the deleted portion of the I_γ2b exon and splice donor site apparently contain sequences necessary for efficient germline gene transcription and thus for CSR to IgG2b.
Keywords:class switch recombination, gene targeting, germline transcripts, Ig

Recent work has shown that the ability of cytokines to direct immunoglobulin heavy chain class-switch recombination to particular heavy chain constant (C) region (CH) genes correlates with the induction of specific germ-line CH transcripts. To test the role of germ-line transcripts in class switching, we have used homologous recombination to mutate the immunoglobulin heavy chain locus of the 18.81A20 murine pre-B-cell line. In the parent cell line, the combination of interleukin-4 (IL-4) and lipopolysaccharide (LPS) induces germ-line epsilon locus transcription prior to class switching to epsilon. The heavy chain locus of the mutated cell line contains the immunoglobulin heavy chain enhancer and variable region gene promoter in place of the LPS/IL-4-responsive germ-line epsilon promoter. The mutant cell line constitutively transcribes the epsilon locus in the absence of IL-4. Strikingly, the mutant cell line also switches to epsilon in the absence of IL-4. This result demonstrates that, at least in the 18.81A20 cell line, germ-line epsilon transcription plays a direct role in class switching to the epsilon locus. In addition, the ability to change the pattern of class switching by altering transcriptional activity indicates that transcription of germ-line CH is mechanistically important in regulation of class switching.

During B cell development V kappa gene rearrangement seems to occur only in mu‐positive pre‐B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre‐B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre‐B cells.

We have isolated a 12 kb clone from the murine genome which we show by DNA transfection studies to contain an entire functional L‐myc gene and the transcriptional promoter sequences necessary for its expression. We have also isolated a 3.1 kb cDNA sequence from a murine brain cDNA library which corresponds to most of the L‐myc mRNA. We have identified the L‐myc coding region within the genomic clone by a combination of S1 nuclease analyses. Northern blotting analyses and comparative nucleotide sequence analyses with the cDNA clone. The L‐myc gene appears to be organized similarly to the other well‐characterized myc‐family genes, c‐myc and N‐myc. The predicted amino acid coding sequence of the L‐myc gene indicates that the L‐myc protein is significantly smaller than c‐ and N‐myc, but is highly related. In particular, comparison of the N‐ and c‐myc protein sequences reveals seven relatively conserved regions interspersed among non‐conserved regions; the L‐myc gene retains five of these conserved regions but lacks two others. In addition, a portion of one highly conserved region is encoded within a different region of the L‐myc gene but, due to changes in the size of L‐myc exons relative to those of N‐ and c‐myc, maintains its overall position in the peptide backbone with respect to other conserved regions. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc‐family proteins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre‐) B cell differentiation, we find that most Abelson murine leukemia virus (A‐MuLV)‐transformed pre‐B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A‐MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre‐B cell lines from normal mice, attempted V kappa‐to‐J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back‐to‐back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site‐specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N‐myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu‐N‐myc genes). High‐level expression of the E mu‐N‐myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu‐N‐myc lines reproducibly developed pre‐B‐ and B‐lymphoid malignancies. The E mu‐N‐myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu‐N‐myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu‐N‐myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N‐myc genes. In contrast, all of the E mu‐N‐myc cell lines had exceptionally low or undetectable levels of the c‐myc RNA sequences, consistent with the possibility that high‐level N‐myc expression can participate in the negative ‘cross‐regulation’ of c‐myc gene expression. Our findings demonstrate that deregulated expression of the N‐myc gene has potent oncogenic potential within the B‐lymphoid lineage despite the fact that the N‐myc gene has never been implicated in naturally occurring B‐lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

The TEL (translocation-Ets-leukemia or ETV6) locus, which encodes an Ets family transcription factor, is frequently rearranged in human leukemias of myeloid or lymphoid origins. By gene targeting in mice, we previously showed that TEL-/- mice are embryonic lethal because of a yolk sac angiogenic defect. TEL also appears essential for the survival of selected neural and mesenchymal populations within the embryo proper. Here, we have generated mouse chimeras with TEL-/- ES cells to examine a possible requirement in adult hematopoiesis. Although not required for the intrinsic proliferation and/or differentiation of adult-type hematopoietic lineages in the yolk sac and fetal liver, TEL function is essential for the establishment of hematopoiesis of all lineages in the bone marrow. This defect is manifest within the first week of postnatal life. Our data pinpoint a critical role for TEL in the normal transition of hematopoietic activity from fetal liver to bone marrow. This might reflect an inability of TEL-/- hematopoietic stem cells or progenitors to migrate or home to the bone marrow or, more likely, the failure of these cells to respond appropriately and/or survive within the bone marrow microenvironment. These data establish TEL as the first transcription factor required specifically for hematopoiesis within the bone marrow, as opposed to other sites of hematopoietic activity during development.

Summary A very sensitive procedure for the determination of baseline levels of platinum is presented. A high-pressure digestion step of the sample is followed by the determination via adsorption voltammetry and detection by a catalytic hydrogen wave. Interferences and blank value problems are discussed.

Little is known about selenium contents in alcoholic beverages, especially in wine, because no reliable determination method was available so far. A procedure for the detection of selenium in the pg- and low ng-range was developed, which uses the hydride-generation-condensation-AAS (HGC-AAS). Selenium contents in 103 German wines of the year vintages 1950 until 1985 were determined. In mean they range between 0.29 and 0.79 µg/l Se and show a certain dependence from the geological soil formation and the wine species. Corresponding vineyard soils from Ingelheim (Rheinhessen, FRG) contain 0.18–0.44 mg/kg Se, mean 0.24 mg/kg Se, 10% of it is available for plants. The distribution of selenium in grape berries shows selenium-rich seeds and — with red wine species — selenium-rich skins. Higher selenium contents by contamination were observed near an industrial location. The behaviour of selenium in the vegetation cycle is pursued. The selenium depletion during fermentation may amount to 60%. All results are summarized in the so-called “trace element vinogram”. Selenium hence is a regular and natural constituent part of wine.

Four methods for the determination of cadmium in blood are compared. In three methods the final step of determination takes place by furnace atomic absorption Spectrometry, in the fourth by anodic stripping voltammetry with a rotating glassy carbon electrode. The sample preparation requires different expense: In the first method a direct determination without matrix separation is accomplished, in the second the protein matrix is precipitated by nitric acid. In methods 3 and 4 the organic matrix is destroyed by an oxidizing digestion, followed in method 3 by an extraction step.

The distribution of molybdato heteropoly acids of phosphorus, arsenic, silicon and germanium as ion pairs with di- and tri-n-octylamine was investigated in dependance of the sulphuric, hydrochloric, nitric and perchloric acid concentration. In the ammonium ion pairs the heteroatom:molybdenum ratio amounts to 1:12, except of the molybdato arsenic acid, where apparently a mixture of the (1:9)- and (1:12)-acid is extracted. The amine:heteropoly acid ratio is variable and depends upon the nature of the mineral acid and the amine. The analytical application for the determination of P, As, Si and Ge was examined. Working instructions for the determination of P and Si in steel are given.

During a period of almost one year airborne dust was collected at the area of ISAS, Dortmund. The total platinum content varied from 0.6 to 130 µg/kg, respectively from 0.02 to 5.1 pg/m3. The impactor measurements resulted in an equal distribution of platinum in combination with particle sizes ranging between 0.5 and 8 µm. The lowest concentrations were observed for the larger particles (>8µm). The “soluble platinum” in airborne dust was determined to be 30 to 43% of the total amount of platinum and 2.5 to 6.9% in tunnel dust.

A procedure is described for the determination of Tl in mineral materials and coal after preconcentration. After dissolution of the sample, the Tl is complexed with dithio-phosphorus acid-O,O-diethylester, ammonium salt, and the complex is adsorbed onto an activated carbon filter. This complexing agent renders it possible to separate the Tl even from strongly acid solutions — and also when Fe is present — up to = 90%. By treating the activated carbon with nitric acid a trace concentrate is obtained practically free of matrices (separation factor > 103), from which the Tl can then be determined without interferences by various spectroscopical methods. When applying ICP/AES (inductively coupled plasma/atomic emission spectrometry) a detection limit of 2 µg/g is obtained; with the usual method of flame-AAS the detection limit is 0.4 µg/g. With the more sensitive graphite furnace technique and the new platinum loop method of flame-AAS, a detection limit of 0.01 µg/g is reached in respect of each of these procedures. By making use of a transient recorder additional information concerning the temporal vaporization behaviour of Tl from the sample matrix can be gained.

To investigate the regulation of lg switch recombination, we have constructed mice with a 56 kb VDJ_μδγ1 transgene. This transgene included an anti-nitrophenyl VDJ segment, S_μ, C_μ, C_δ, I_γ1, S_γ1, C_γ1 and the C_γ1 membrane exons from the murine Igh^a haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of C_μ (which is >95% transgenic), C_δ and other cell surface markers, and normal amounts of VDJ and C_μ RNA. γ1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS+IL-4 or CD40L+IL-4. B cells from both lines of transgenic mice expressed transgenic γ1^a after in vitro culture with CD40L+IL-4, but not after culture with CD40L alone. However, the CD40L+IL-4 induced IgG1 precursor frequency is much lower for VDJ_μδγ1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

The IgH locus intronic enhancer (E_μ, located in the intron between the J_H segments and the C_μ gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of E_μ, plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neo^r gene and analyzed the switch status of the mutated IgH loci. E_μ/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neo^r gene replacing the E_μ/MAR showed relatively normal switch frequency. These findings indicate that the E_μ/MAR region plays a significant, but not necessary role in facilitating class switching at the μ locus. Potential mechanisms for these findings are discussed.
Keywords:class switching, gene targeting, IgH intronic enhancer, matrix attachment regions

To investigate potential roles of the RAG-1 and RAG-2 gene products in Ig heavy chain class recombination (CSR), we have generated RAG-1^-/- and RAG-2^-/- mice which contain both a rearranged IgHC V(D)J gene (referred to as B1-8) inserted into the endogenous Ig heavy chain (HC) locus in place of the J_H segments, and a rearranged δ1 light chain (LC) transgene (which are referred to as RAG-1^-/-B1-8δ and RAG-2^-/-B1-8δ mice respectively). The B1-8 HC gene and δ LC genes encode proteins that associated to form a complete Ig molecule, the expression of which leads to substantial reconstitution of the peripheral B cell compartments of RAG-1^-/-B1-8δ and RAG-2^-/-B1-8δ mice. Both RAG-1^-/-B1-8δ and RAG-2^-/-B1-8δ mice have relatively normal levels of the various IgG isotypes, but greatly reduced levels of serum IgM and IgA compared to normal littermates. Furthermore, RAG-1^-/-B1-8δ and RAG-2^-/-B1-8δ B cells activated in vitro with lipopolysaccharide (LPS) or LPS plus IL-4 responded similarly to control B cells with respect to surface expression and secretion of IgG3, IgG1, IgG2b, IgG2a and IgE, but again were deficient in the secretion of IgM. Together, these findings indicate that neither RAG-1 nor RAG-2 expression is required for efficient class switching to most HC isotypes in B cells.

Defective expression of the Ku80 gene has been implicated as underlying the V(D)J recombination and DNA double-strand break repair defects in the xrs-6 Chinese hamster ovary cell line. We now show that the mutation in the Ku80 gene involves a G to A transition 15 bp upstream of exon 2. This mutation creates a new splice acceptor site which results in the generation of Ku80 transcript that cannot encode a functional product due a 13 nucleotide insertion and a resulting frameshift.

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

We have previously demonstrated a dramatic preference for utilization of the most JH-proximal VH gene segments in the newborn liver versus adult spleen. We now examine in detail the relative expression of different VH gene families throughout ontogeny and in immunodeficient mice to gain insight into factors that cause the shift in VH usage. We find that the relative expression of VH gene families remains constant and biased throughout fetal and neonatal liver development. In addition, the primary VH repertoire expressed in neonatal spleen displays a similarly biased, position-dependent VH repertoire. The pattern of VH gene expression begins to change at 5-7 d postnatally and reaches the adult randomized pattern at approximately 2 wk of age. We also find biased expression of JH-proximal VH gene families in adult bone marrow and in spleens of adult leaky scid mice, suggesting that the spontaneously generated repertoire of adult mice is similar to that observed in neonates. Together, these data suggest that a position-dependent repertoire is generated in differentiating pre-B cells at all stages of ontogeny, at least in part, as a result of preferential rearrangement of proximal VH gene segments. Therefore, mechanisms subsequent to V gene rearrangement, such as regulatory interactions and antigen selection, must play a major role in normalizing the repertoire.

Transgenic mice that contain constructs of the L-myc gene under the transcriptional control of the immunoglobulin heavy chain enhancer (E mu) develop thymic hyperplasia and are predisposed to T cell lymphomas. Here we describe a second form of malignancy that occurs in aging E mu L-myc transgenic mice. The mean latency period for the development of this malignancy is longer compared with the E mu L-myc T cell lymphomas but the overall incidence is increased threefold. The histopathological morphology is that of a highly malignant mesenchymal neoplasm that closely resembles human fibrous histiocytoma. The tumor cells were classified as myelomonocytic on the basis of several lineage-specific markers and the lack of rearrangements of the immunoglobulin heavy chain and the T cell receptor beta loci. Cultured tumor cells produce macrophage colony-stimulating factor (M-CSF) protein and express the M-CSF receptor, suggesting the involvement of an autocrine loop in this malignancy. Similar to the E mu L-myc T cell lymphomas, these tumors show high-level transgene expression but no detectable levels of endogenous c-myc mRNA, directly implicating the deregulated expression of L-myc in the generation of this malignancy. E mu L-myc myelomonocytic tumors show consistent trisomy of chromosome 16, implicating this as a secondary event in the development of this tumor. In the light of recent findings that L-myc is expressed in human myeloid leukemias and in several human myeloid tumor cell lines, the results described here might implicate L-myc in the development of naturally occurring myeloid neoplasias.

We have tested the ability of the T cell receptor beta (TCR-beta) transcriptional enhancer (E beta) to confer transcriptional activation and tissue-specific V(D)J recombination of TCR-beta V, D, and J segments in a transgenic minilocus recombination substrate. We find that the minimal E beta element, as previously shown for a DNA segment that contained the E mu element, promotes a high level of substrate D to J beta rearrangement in both B and T cells, but only promotes V beta to DJ beta rearrangement in T cells. Thus, both the E mu and E beta elements similarly direct V(D)J recombination of this substrate in vivo, supporting a general role for transcriptional enhancers in the normal regulation of this rearrangement process. Surprisingly, however, we found that both the V beta and DJ beta portion of the constructs were transcribed in an enhancer-dependent fashion (conferred by either E mu or E beta) in both B and T lineage cells, including normal precursor B cells propagated in culture. These findings indicate that, at least in some contexts, transcriptional activation, per se, is not sufficient to confer V(D)J recombinational accessibility to a substrate V gene segment.

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.

N-myc, a cellular gene bearing homology to the c-myc protooncogene, is frequently amplified and overexpressed in a highly restricted set of related tumors, most notably neuroblastomas and retinoblastomas. We have examined the possibility that N-myc may play a causal role in the genesis of these tumors by defining its ability to transform primary cells in tissue culture. Using an N-myc expression construct capable of producing constitutively deregulated levels of full-length murine N-myc mRNA, we demonstrate that a deregulated N-myc gene can cooperate with the activated Ha-ras oncogene to cause tumorigenic conversion of normal embryonic fibroblasts in a manner indistinguishable from the deregulated c-myc oncogene. Cell lines established from N-myc/ras-transformed foci express high levels of the N-myc gene, and such lines are similar to c-myc/ras transformants in their ability to grow in soft agar and cause tumors in syngeneic rats. These results illustrate that N-myc does encode a c-myc-like transforming activity and that this transforming activity is not specific for the very restricted set of tumors in which N-myc is normally amplified or overexpressed.