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Cell Growth & Differentiation Vol. 13, 39-45, January 2002
© 2002 American Association for Cancer Research

Hmgi-c-independent Activation of MuRantes in Vivo

John F. Schiltz, Krishna Kesari, Hena R. Ashar and Kiran Chada1

Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The architectural factor HMGI-C is of considerable interest for its recognized roles in mammalian development and tumorigenesis. As a result, the identification of downstream target genes of HMGI-C is the present focus of active research. In vitro evidence from macrophage cell lines has previously suggested that Hmgi-c is necessary for the inducible activation of MuRantes expression. To attempt to verify this hypothesis, an in vivo analysis was performed that took advantage of the existence of the Hmgi-c null mouse strain. The ability of cells and tissues extracted from Hmgi-c null mice to express the inflammatory chemokine MuRantes was investigated. The investigation examined MuRantes expression in primary embryonic fibroblasts and fresh peritoneal macrophages after Newcastle disease virus induction and whole organs after lipopolysaccharide induction. Each of these systems clearly demonstrates that Hmgi-c is not required for the activation of MuRantes expression.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The HMGI protein family has been extensively studied for its involvement in development and cancer (1) . It consists of three members, HMGI and HMGI(Y) (alternately spliced products of a single HMGI(Y) gene) and HMGI-C (the single product of the HMGI-C gene; Refs. 2, 3, 4 ). Structurally, the HMGI family proteins are each composed of three highly conserved DNA binding domains termed AT-hooks because they specifically recognize AT-rich sequences of DNA (5) . These motifs specifically bind within the minor groove of DNA and induce conformational changes in the DNA structure that allow for transcription factor binding (6) . They also possess a highly acidic COOH-terminal domain that can be both acetylated and phosphorylated and that modulates their function (7, 8, 9) . The best characterized biochemical function for an HMGI protein is the requirement for HMGI(Y) during the NF2 -{kappa}B-dependent activation of human IFN-ß expression by its virus-inducible enhancer (10) . HMGI(Y) functions as an integral component of the enhanceosome both by contributing to its stability and by recruiting additional transcription activators to the promoter (11 , 12) . Although its involvement is not as well described, HMGI-C has also been shown to enhance the transcription of IFN-ß induced by NF-{kappa}B, and because of the structural similarities between HMGI-C and HMGI(Y), HMGI-C has been postulated to act by a similar method of action (13) .

In humans, HMGI-C is predominantly expressed during the embryonal and fetal periods of development (14) . Similarly, in the mouse, highest levels of Hmgi-c RNA are detectable from 10.5 dpc to 13.5 dpc, after which, expression falls off dramatically, being undetectable (by Northern) by 16.5 dpc (15) . Hmgi-c has not been detectable in a variety of newborn and adult tissues (14 , 16 , 17) . A targeted disruption of the murine Hmgi-c gene results in mice that are small in stature (about 40% the weight of wild-type mice), with most of their organs proportionately reduced in size, a phenotype measurable by 15.5 dpc (15 , 18) . The normal expression of HMGI-C in both mice and humans is predominantly restricted to cells of the developing mesenchyme during early development (14 , 15) . It is thus believed that Hmgi-c is necessary for normal organogenesis (of predominantly mesenchymal tissues) prior to their overt differentiation (15) .

It is well established that HMGI expression is associated with aberrant states of cellular proliferation and differentiation (3 , 19, 20, 21) . In mice, tumors induced by viral, chemical, and cellular means express high levels of each of the Hmgi genes (22) . Also, the transformation of rat thyroid cells has been shown to require Hmgi-c, because antisense Hmgi-c expression blocks viral transformation (23) . In humans, translocations that disrupt HMGI-C and result in misexpression of fusion, truncated and normal transcripts of the gene are found in a high percentage of benign mesenchymal tumors including lipomas, pulmonary chondroid hamartomas, uterine leiomyomas, and endometrial polyps (24, 25, 26, 27) . Because HMGI-C expression is not normally detected in these differentiated cell types, the specific misexpression of the HMGI-C gene is believed to be a primary cause of their pathogenesis.

RANTES is a 68-amino-acid, Mr 8,000 chemokine that is the prototype member of the C-C subgroup of the Scya superfamily of chemokines (28) . Although primarily expressed by T cells, RANTES is also found in a variety of different cell types including isolated peripheral blood leukocytes (after induction with antigen or growth factors) and the mouse macrophage cell line RAW 264.7 (29, 30, 31) . RANTES is selectively chemotactic for monocytes, eosinophils, and the memory-helper subset of T-lymphocytes (32 , 33) . The Scya5 gene encodes the murine homologue of RANTES (MuRantes) that is 85% identical (in amino acid sequence) to the human protein (29 , 31) . MuRantes is selectively chemotactic for human monocytes which demonstrates that its activity is somewhat species nonspecific (31) . The inflammatory cell chemotactic activity of RANTES has been implicated as an important component in the pathogenesis of a variety of human diseases including atherosclerosis, asthma, allergic rhinitis, and organ transplant rejection (34, 35, 36, 37, 38) .

In adult tissues, RANTES expression can be detected in inflamed tonsils and normal spleen, lung, and kidney (39 , 40) . Unexpectedly, RANTES expression is also found in human fetal tissues and some well-differentiated tumor types/cell lines (40) . Fetal tissues shown to express RANTES include the developing lung, thymus, spleen, and kidney (40 , 41) . Expression in the developing kidney depends on differentiation state, because the least differentiated cells of the subcapsular nephrogenic blastema (the outermost area of the developing kidney) show the highest levels of RANTES mRNA (in situ hybridization data), whereas expression decreases dramatically with increasing differentiation toward the center of the organ (40) . This differential expression as a function of differentiation state points to a possible role for RANTES in the development of tissues. Interestingly, this pattern of expression is comparable with that observed for Hmgi-c in the developing mouse kidney,3 allowing for the possibility that two genes are associated during development.

A report has suggested a link exists between Hmgi-c and MuRantes (42) . Hmgi-c was shown to bind to the virus response element (VRE) of the MuRantes promoter from NDV-stimulated nuclear extracts of the RAW 264.7 macrophage cell line. It also demonstrated that alteration of two AT-rich regions of the promoter (putative HMGI binding sites) ablated both the association of Hmgi-c with the promoter as well as the inducible promoter activity (42) . This was interpreted to mean the association of the Hmgi-c protein with the MuRantes promoter is required for the normal activation of MuRantes. A LPS response element has also been characterized within the RANTES promoter (43) . A similar, but less pronounced effect was described using LPS-stimulated extracts (42) . The existence of the Hmgi-c null mice allowed for further examination of this implied requirement. The physiological models allowed for the unique opportunity to test this requirement in an in vivo system. Herein we describe the induction of MuRantes in primary embryonic fibroblasts, peritoneal macrophages, and whole adult tissues extracted from Hmgi-c-/- mice.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Developmental Expression of MuRantes.
A Northern analysis was performed to examine the endogenous expression pattern of MuRantes during development (Fig. 1A)Citation . No expression was detected in the RNA isolated from embryos, which indicated that the level of MuRantes during development is below the assay detection limits. The same blot was subsequently analyzed using Hmgi-c- and Hmgi(y)-specific probes, and the embryonic RNAs demonstrated the expected expression pattern, decreasing sharply as development proceeds. To detect the previously reported (40) expression of MuRantes during development, a more sensitive, MuRantes-specific RT-PCR analysis was performed on RNA extracted from 12.5, 14.5, and 16.5 dpc wild-type and Hmgi-c-/- embryos (Fig. 1B)Citation . A product of the appropriate size (337 bp) was visualized in each sample examined. This demonstrates that, during development, Hmgi-c is not required for MuRantes expression. To test the fidelity of each RNA sample, RT-PCR was performed analyzing Gapdh expression, and each sample of RNA demonstrated the expected 436 bp band.



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  		Fig. 1. A, developmental expression pattern of MuRantes, Hmgi-c, and Hmgi(y). Numbers 10–18, RNA extracted from embryos of 10.5 dpc through 18.5 dpc. M, mock infected wild-type embryonic fibroblasts; N, NDV-infected wild-type embryonic fibroblasts. Three µg of each RNA sample were applied to the gel, and equal loading was verified using a 28S rRNA-specific probe. Approximate sizes of transcripts are 1.1 kb (MuRantes); 4.1 kb (Hmgi-c); 1.7 kb (Hmgi(y)) and 4.8 kb (28S rRNA). B, RT-PCR analysis of RNAs from wild-type and Hmgi-c-/- embryos. 12.5, 14.5, and 16.5: ages in dpc of analyzed embryos. M, marker (1-kb DNA ladder); W, wild-type embryo; N, Hmgi-c null embryo; L, adult mouse lung; X, water. Sizes of bands are 337 bp (MuRantes) and 436 bp (Gapdh).

 
Induction of MuRantes in Hmgi-c-/- Fibroblasts and Macrophages.
Wild-type and Hmgi-c-/- primary embryonic fibroblasts (15) were isolated from 13.5-dpc embryos. RNA extracted from these cultures was examined by Northern analysis after overnight induction by NDV. Fibroblasts of both genotypes were equally able to express MuRantes after NDV infection of the cultures (Fig. 2)Citation . Thus, there is no requirement for embryonic fibroblasts for Hmgi-c to express MuRantes. Wild-type and Hmgi-c-/- macrophages, freshly isolated from the peritoneum of starch-injected mice, were similarly analyzed. These cells were chosen because primary peritoneal macrophage cultures are known to express MuRantes after NDV induction (42) . The wild-type macrophages demonstrated exceptionally high levels of expression of MuRantes (Fig. 2)Citation . Similar high levels of MuRantes expression were also observed in the Hmgi-c-/- macrophages. Thus, once again, an absence of Hmgi-c had no affect on the inducible expression of MuRantes.



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  		Fig. 2. The MuRantes induction in primary embryonic fibroblasts (Fibroblasts), freshly isolated peritoneal macrophages (Macrophages), and 13.5 dpc embryos is shown. Mock and NDV, the split cell cultures were infected with virus (NDV) or only received uninfected allantoic fluid (Mock) prior to a 24-h incubation. One µg of each sample was applied to the gel, and equal loading was again verified using a 28S rRNA-specific probe. WT, wild-type cells; N, Hmgi-c-/- cells. Approximate sizes of transcripts are 1.1 kb (MuRantes) and 4.8 kb (28S rRNA).

 
Induction of Hmgi Genes.
The levels of Hmgi RNA were subsequently analyzed in the same fibroblast and macrophage samples. As expected, both Hmgi-c and Hmgi(y) were expressed in the primary embryonic fibroblasts (Fig. 3)Citation . Interestingly, Hmgi(y) was induced in both wild-type and Hmgi-c-/- fibroblasts after NDV induction, whereas Hmgi-c was not induced. The freshly isolated macrophages demonstrated a low level of Hmgi(y) and no Hmgi-c expression. Hmgi(y) was also induced in macrophages after NDV infection.



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  		Fig. 3. The induction of the Hmgi genes in primary embryonic fibroblasts (Fibroblasts), freshly isolated peritoneal macrophages (Macrophages), and 13.5-dpc embryos is shown. For labels, refer to Fig. 2Citation . One µg of each sample was applied to the gel, and equal loading was again verified using a 28S rRNA-specific probe. Approximate sizes of transcripts are 4.1 kb (Hmgi-c); 1.7 kb [Hmgi(y)]; and 4.8 kb (28S rRNA).

 
Induction in LPS-induced Tissues.
To broaden the scope of this study, LPS stimulation in whole tissues was used as a second system for the induction of MuRantes.

LPS was injected into the peritoneum of adult (3-month-old) wild-type and Hmgi-c-/- mice to look at MuRantes induction in whole tissues. Twenty-four h after the injection, tissues were perfused with PBS, and RNA was isolated from the lung, kidney, and spleen. Northern analysis on 3 µg of total RNA revealed low-level MuRantes expression in wild-type and Hmgi-c-/- lung and spleen without LPS stimulation (Fig. 4)Citation . After LPS injection, MuRantes was significantly induced in lung, kidney, and spleen regardless of genotype. No expression of Hmgi-c was observed in any of the adult tissues, consistent with previous reports (17) . A minor induction of Hmgi(y) was observed in the LPS-induced tissues. Even with this second inducing stimulus, Hmgi-c was shown not to be required for MuRantes induction.



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  		Fig. 4. The induction of MuRantes and the Hmgi genes is shown after LPS stimulation of adult tissues. Tissues were extracted from wild-type and Hmgi-c null mice. Saline, a saline solution was injected (negative control); LPS, a LPS solution was injected. WT Fib., wild-type fibroblasts. Three µg of each sample were applied to the gel, and equal loading was again verified using a 28S rRNA-specific probe. Approximate sizes of transcripts are 1.1 kb (MuRantes); 4.1 kb (Hmgi-c); 1.7 kb [Hmgi(y)], and 4.8 kb (28S rRNA).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The activation of expression of the MuRantes gene after induction in a macrophage cell line has been previously reported to require Hmgi-c (42) . An examination of the induction of MuRantes in cells from Hmgi-c null mice (fibroblasts, macrophages, and whole tissues) was used to analyze the hypothesis that the genes are in the same pathway. Our experiments clearly indicate that Hmgi-c is not required for the NDV- or LPS-inducible expression of MuRantes in embryonic fibroblasts, peritoneal macrophages, or whole adult tissues.

The normal expression pattern of MuRantes was examined in wild-type and Hmgi-c null mice during development. RT-PCR analysis demonstrated low-level MuRantes RNA in embryo extracts. The MuRantes signal was detectable in both wild-type and Hmgi-c null embryo RNAs. Thus, there is no requirement for Hmgi-c for the endogenous developmental expression of MuRantes.

This did not entirely rule out the possibility that the two genes are in a common pathway, however, because the reported interaction between Hmgi-c and MuRantes occurred with a high-level of MuRantes expression in a macrophage cell line after viral induction (42) . To test the inducibility of cells for MuRantes expression, both wild-type and Hmgi-c-/- primary embryonic fibroblasts isolated from 13.5-dpc embryos were cultured. Twenty-four h after infection with NDV, both genotypes demonstrated obvious MuRantes induction. These data indicate that Hmgi-c is not required for the inducible expression of MuRantes.

Freshly isolated peritoneal macrophages are the most physiologically relevant cellular system to respond to a viral stimulus and to express MuRantes. Consistent with this, the macrophages demonstrate the greatest MuRantes response to NDV induction. As was observed with embryonic fibroblasts, the Hmgi-c-/- macrophages were not attenuated in their ability to express MuRantes after NDV induction. Finally, the ability of LPS to induce MuRantes in whole adult tissues was also shown to be independent of the presence of Hmgi-c. Thus, the in vivo systems examined in the present study showed no indication that Hmgi-c is required for MuRantes. Neither the normal, low-level MuRantes expression found during development nor the inducible, high-level expression resulting from two different induction stimuli requires Hmgi-c.

There is still the possibility that Hmgi-c normally plays a role in MuRantes expression but that its absence is completely compensated for by other factors in the Hmgi-c null mice. Biochemically, for the homologous HMGI family member Hmgi(y), Hmgi-c can be substituted to enhance the activity of the transcription factor NF-{kappa}B (13) . However, the in vivo evidence suggests that no functional compensation exists between the two genes. For example, the expression levels of Hmgi(y) are not altered in the absence of Hmgi-c, and the normal expression of Hmgi(y) does not correct the small phenotype of the Hmgi-c null mice (15) . Secondly, Hmgi-c null mice resist weight gain caused by obesity-inducing stimuli even though they express Hmgi(y) (44) . In fact, the absence of Hmgi-c prevents the proliferation of adipocytes (44 , 45) , whereas the suppression of Hmgi(y) results in an increased growth rate and impaired differentiation of the adipocytes (45) . Thus, it has been suggested that Hmgi-c may counteract Hmgi(y) as seen during adipocyte proliferation and differentiation (45) .

The most obvious explanation of the apparent discrepancy between results presented here and those suggesting an association between Hmgi-c and MuRantes is simply that the requirement is not consistent between the different systems. The RAW 264.7 cells are a transformed (by Abelson leukemia virus) murine macrophage cell line that has been cultured for more than 20 years (46) . None of the systems used in this study require the establishment of a transformed cell type, and each one is, thus, more physiologically relevant than the RAW 264.7 cell line. The very fact that Hmgi-c is present in the RAW 264.7 cell line is actually a significant aberration, for Hmgi-c is neither expressed nor induced in freshly isolated macrophages. Thus, the observation that MuRantes is dependent on Hmgi-c is likely a culture-system-specific artifact.

The description of the NDV inducible expression of Hmgi(y) in fibroblasts and macrophages is the first such report. Importantly, this result provides unique and potentially useful new systems for the future study of the induction of Hmgi(y). The new systems could be used to identify new important genes in a common in vivo pathway with Hmgi(y). The induction of Hmgi(y) by LPS has been previously demonstrated (47) . Additionally, the coincident induction of Hmgi(y) and MuRantes in each of the systems has not been overlooked and may indicate that it is actually these two genes that are in a common pathway. The RANTES promoter is known to be up-regulated in cell culture by NF-{kappa}B (48) , the same transcription factor whose downstream activation of IFN-ß is known to require HMGI(Y) (10) . Obviously additional experiments are required to demonstrate whether any sort of a requirement exists of the MuRantes promoter for Hmgi(y).

The present analyses demonstrate that no physiologically relevant requirement exists of MuRantes for Hmgi-c. The many roles played by the HMGI gene family in mammalian development and mesenchymal tumorigenesis underscores the importance of identifying downstream factors. This process is an important next step in understanding the roles of the protein in development and cancer. Importantly, downstream target genes could be additional targets for clinical intervention in the vast array of tumors associated with Hmgi-c misexpression. However, the consistent expression of MuRantes in Hmgi-c null cells makes it unlikely that the two genes share any such common pathway in vivo.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Wild-type and Hmgi-c Null Embryo RNA Isolation.
The generation of mice with a targeted disruption in the Hmgi-c gene has been described previously (15) . Heterozygous male and female mice were mated to produce embryos of the desired ages. Noon of the morning when a copulatory plug is observed is considered 0.5 dpc. Pregnant mice with embryos of the required gestational ages were killed, and the individual embryos isolated. For genotyping, the embryo-derived amnions were saved for high-molecular-weight DNA extraction by a standard protocol (49) . The wild-type and disrupted Hmgi-c alleles were amplified by PCR in a protocol described previously (44) . RNA from embryos was isolated using a cesium chloride method described previously (50) .

Isolation and Culture of Embryonic Fibroblasts.
Embryos (13.5 dpc) were isolated from pregnant Hmgi-c+/- mice and placed into sterile PBS. After removal of their heads and visceral organs, the remains of the embryos were minced in a 2-ml solution of 0.005% trypsin and incubated in this solution for 10 min at 37°C. DMEM (10 ml; with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin) was then added and mixed to quench the enzyme. The large embryo debris was then allowed to settle, and the supernatant containing embryonic fibroblasts was plated into 10-cm dishes and cultured at 37°C (in 5% CO2). After one passage, duplicate plates of wild-type and Hmgi-c-/- fibroblasts were infected with NDV as described below.

Isolation and Culture of Peritoneal Macrophages.
Wild-type and Hmgi-c-/- mice were injected i.p. with 40 µl of a sterile 2% starch solution per gram body weight. Three days later, the mice were killed, and their peritoneal cavities were flushed with cold PBS. Ten wild-type and 20 Hmgi-c null mice were required to obtain sufficient numbers of macrophages for culture. The PBS that contained macrophages was then centrifuged, and the cell pellet was mixed with DMEM (with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin) and placed into culture at 37°C (in 5% CO2). The following day, duplicate monolayer plates of wild-type and Hmgi-c-/- macrophages were infected with NDV as described below.

NDV Infection of Cultured Macrophages and Fibroblasts.
Monolayers of cultured macrophage and fibroblast cells were infected with NDV (NDV, LaSota strain; Charles River Laboratories) as follows. Plates were first washed with PBS, and thereafter 1 ml of DMEM (without serum or antibiotics) with 100 µl of NDV-infected allantoic fluid (>1 x 109 EID50/ml) were added. As controls, duplicate plates received DMEM with 100 µl of uninfected allantoic fluid. Cells were incubated for 1 h at 37°C and then washed with PBS. Plates were then incubated in DMEM (with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin) for 24 h at 37°C, after which RNA was isolated using the TRIzol reagent according to the manufacturer’s protocol (Life Technologies, Inc.).

LPS Induction and Dissection of Adult Tissues.
Wild-type and Hmgi-c-/- mice of the same age and sex (male) were injected with either a sterile solution of 0.85% saline or a 10 mg/ml solution of LPS (Escherichia coli 0111:B4) in 0.85% saline as described previously (51) . LPS (100 µg) was injected per gram of body weight into the mice, and for the control animals, an equivalent volume of saline was injected. Twenty-four h postinjection, animals were perfused with PBS through their left ventricles, and the tissues were harvested once the lungs were completely blanched. Lung, kidney, and spleen were dissected, and RNA was isolated using a cesium chloride method described previously (50) .

MuRantes Expression by RT-PCR.
Reverse transcription was performed using 1 µg of total RNA. Fifty pmol of a MuRantes-specific antisense (reverse) primer from exon 3 (no. 8297: 5'-CATGCCATGGTACAGGGTCAGAATCAAG-3') were mixed with the RNA in a 12.5-µl total volume. The mix was heated to 70°C for 10 min, and then quickly placed on ice. Then 4 µl of 5x first strand buffer [1 = 50 mM Tris-HCl (pH 7.5), 75 mM KCl, 3 mM MgCl2], 2 µl of 0.1 M DTT, 0.5 µl of 20 mM dNTP mix, and 1 µl (200 units) of Superscript II RT were added and the reaction was placed at 42°C for 1 h. The enzyme was subsequently inactivated with a 15-min incubation at 70°C. Two µl of the transcribed cDNA was then used in a 50-µl PCR reaction [10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 50 mM KCl, 0.4 mM each dNTP) with 2.5 units Taq DNA polymerase, 50 pmol of reverse primer no. 8297 and 50 pmol of a MuRantes-specific sense (forward) primer from exon 1 (no. 8298: 5'-GGAATTCCTGCCGCGGGTACCATGAAG-3'). After a 5-min denaturation at 94°C, 35 cycles of amplification (94°C for 30 s, 54°C for 30 s, and 72°C for 1 min) were performed followed by a 7-min final extension at 72°C. The resulting 337-bp product encompassed the entire coding region of the gene and was resolved by gel electrophoresis in a 2% agarose gel and visualized with ethidium bromide staining. As a control for the fidelity of the RNA, samples were also tested for the presence of the GAPDH transcript. Reverse transcription and PCR reaction conditions were exactly the same as they were for the MuRantes protocol, except the GAPDH-specific reverse (5'-CTAAGCAGTTGGTGGTGCAGG-3'; from exon 7) and forward (5'-CGTATTGGGCGCCTGGTCAC-3'; from exon 2) primers were used with a 60°C annealing temperature during the 35 cycles of amplification. Again, the product (435 bp) was resolved by gel electrophoresis in a 2% agarose gel and visualized by ethidium bromide.

Northern Hybridization.
Total RNA (1–3 µg) was resolved by electrophoresis and transferred to a nylon membrane (Duralon UV; by Stratagene). The probes for Northern analyses were all generated by random primed labeling of the probe DNA fragments (as described in the following section). Hybridization and wash conditions were essentially as described previously (51) . Reprobing of blots was accomplished by first stripping the blots with several changes of stripping solution [1% SDS, 0.1x SSC and 50 mM Tris-HCl (pH 7.5)] at 80°C for a period of 2 h (until clean).

Generation of Probe DNA.
The MuRantes specific probe was generated by the cloning of a RT-PCR product into the pCR2.1-TOPO cloning vector according to the manufacturer’s (Invitrogen) instructions as follows. MuRantes-specific RT-PCR (described earlier in the "Materials and Methods") was used to generate a 337-bp product from adult mouse lung total RNA. The resulting large plasmid prep (pCR2.1RANTES) was sequenced and shown to contain the MuRantes insert. The plasmid was then digested with EcoRI, and the 340-bp DNA fragment that was generated was isolated after separation by gel electrophoresis and extraction from the agarose gel using GeneCleanII according to the manufacturers protocol. This fragment was subsequently labeled and used in Northern analyses. The 28S rRNA-specific human DNA probe was also made by cloning a RT-PCR product into the pCR2.1-TOPO vector. The 28S-specific cDNA was transcribed from 13.5-dpc embryonic RNA with a reverse primer (5'-GAAGAGCCGACATCGAAGG-3'). The transcribed cDNA was then used in a PCR reaction with the 28S-specific reverse primer (above) and a 28S rRNA-specific forward primer (5'-ACAGTGCCAGGTGGGGAGTTTG-3'). The PCR reaction conditions were identical to those used for the MuRantes RT-PCR, except that the annealing temperature was 60°C. The resulting plasmid was amplified, purified, and sequenced. The 28S fragment was separated by gel electrophoresis from the vector after digestion with EcoRI, and the insert was purified using the QIAquick Gel extraction system according to the manufacturer’s instructions.

Both of the Hmgi probes were described previously (15) . When isolated from their plasmids, the Hmgi-c-specific probe fragment consists of exons 2 and 3 of the murine Hmgi-c gene, whereas the Hmgi(y)-specific probe fragment is composed of the entire coding region of the mouse gene.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Biochemistry, University of Medicine and Dentistry, Piscataway, NJ 08854. Phone: (732) 235-4768; Fax: (732) 235-3965; E-mail: chada{at}umdnj.edu Back

2 The abbreviations used are: NF, nuclear factor; dpc, day(s) post coitum; RANTES, (chemokine) regulated upon activation normal-T-cell-expressed and -secreted; NDV, Newcastle disease virus; LPS, lipopolysaccharide; RT-PCR, reverse transcription-PCR. Back

3 J. F. Schiltz, K. F. Benson, and K. Chada, unpublished observations. Back

Received for publication 9/ 5/01. Revision received 11/ 7/01. Accepted for publication 11/13/01.


   References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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