The bona fide mouse U7 snRNA gene maps to a different chromosome than two U7 pseudogenes.

The U7 snRNA, together with both common and unique snRNP proteins, forms the U7 snRNP particle. This particle is a major component of the 3' processing machinery that converts histone pre-mRNA into mature mRNA in the eukaryotic nucleus. The genes for many snRNAs are present in multiple copies and often have many pseudogenes. Southern blot experiments using U7 oligonucleotide and gene probes have identified only one strongly hybridizing band and three weakly hybridizing bands in mouse genomic DNA. Previously, two laboratories isolated genomic clones encoding one functional U7 gene and three presumed pseudogenes. Since all the genes were isolated on separate, nonoverlapping genomic fragments, the four genes are not tightly clustered in the mouse genome. In this study, we use fluorescence in situ hybridization to determine the chromosomal locations of these clones and their possible linkage to histone loci. Two of the pseudogenes map to mouse Chromosome 1, but are many megabases apart, whereas the active U7 gene maps to Chromosome 6. Possible mechanisms for this localization pattern are discussed.

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus.

Cloning of a full-length cDNA encoding bovine interleukin 4 by the polymerase chain reaction

A human interleukin 4 (hIL-4)-encoding cDNA (hIL4) probe was used to screen a bovine genomic library, and three clones containing sequences with homology to the human and mouse IL4 cDNAs were isolated. Sequence information obtained from one of these genomic clones was used to design an oligodeoxyribonucleotide primer corresponding to the transcription start point region for use in the polymerase chain reaction (PCR). The PCR-RACE protocol, designed for the rapid amplification of cDNA ends, was successfully used to generate a full-length bovine IL4 (bIL4) cDNA clone from polyadenylated RNA isolated from concanavalin A-stimulated bovine lymph node cells. The bIL4 cDNA is 570 bp in length and contains an open reading frame of 405 nucleotides (nt), coding for a 15.1-kDa precursor of 135 amino acids (aa), which should be reduced to 12.6 kDa for unglycosylated bIL4 after cleavage of a putative hydrophobic leader sequence of 24 aa. The aa sequence contains one possible Asn-linked glycosylation site. Bovine IL4 is shorter than mouse (mIL4) and hIL4, because of a 51-nt deletion in the coding region. Comparison of the overall nt and deduced aa sequences shows a greater homology of bIL4 with hIL4 than with mIL4. This homology is not evenly distributed, however, with the nt sequences 5' and 3' of the coding region showing a much greater homology between all three species than the coding sequence.

Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

Chlorophyll catabolism and gene expression in the peel of ripening banana fruits

The biochemical and molecular basis of chlorophyll (Chl) catabolism in bananas was investigated during ripening at 20°C and at an elevated temperature (35°C) where degreening is inhibited. Biochemical analysis showed that Chl breakdown products could be isolated from fruit ripened at both temperatures. The coloured breakdown products, chlorophyllide and pheophorbide, were not detected at any stage of ripening in the two treatments; however, a non-fluorescent Chl catabolite accumulated to a higher concentration at 20 than at 35°C. To investigate the ripening-related gene expression associated with these changes, a cDNA library was generated from the peel of fruit ripened at 20°C. Differential screening of this library produced 20 non-redundant families of clones including those encoding enzymes involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation and other metabolic events. The expression of these genes was followed by northern analysis in fruit ripened at 20 and 35°C.