Several genes are found on both the human X and Y chromosomes in regions that do not recombine during male meiosis. In each case, nucleotide sequence analysis suggests that these X-Y gene pairs encode similar but nonidentical proteins. Here we show that the human Y- and X-encoded ribosomal proteins, RPS4Y and RPS4X, are interchangeable and provide an essential function: either protein rescued a mutant hamster cell line that was otherwise incapable of growth at modestly elevated temperatures. These findings are consistent with the hypothesis that RPS4 deficiency has a role in Turner syndrome, a complex human phenotype associated with monosomy X.

Several genes are found on both the human X and Y chromosomes in regions that do not recombine during male meiosis. In each case, nucleotide sequence analysis suggests that these X-Y gene pairs encode similar but nonidentical proteins. Here we show that the human Y- and X-encoded ribosomal proteins, RPS4Y and RPS4X, are interchangeable and provide an essential function: either protein rescued a mutant hamster cell line that was otherwise incapable of growth at modestly elevated temperatures. These findings are consistent with the hypothesis that RPS4 deficiency has a role in Turner syndrome, a complex human phenotype associated with monosomy X.

A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell line after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 degrees C were shifted to 39.5 degrees C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in G1 phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10(-3). On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA.

A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell line after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 degrees C were shifted to 39.5 degrees C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in G1 phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10(-3). On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA.

Several genes are found on both the human X and Y chromosomes in regions that do not recombine during male meiosis. In each case, nucleotide sequence analysis suggests that these X-Y gene pairs encode similar but nonidentical proteins. Here we show that the human Y- and X-encoded ribosomal proteins, RPS4Y and RPS4X, are interchangeable and provide an essential function: either protein rescued a mutant hamster cell line that was otherwise incapable of growth at modestly elevated temperatures. These findings are consistent with the hypothesis that RPS4 deficiency has a role in Turner syndrome, a complex human phenotype associated with monosomy X.

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.

Reverse-phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4 reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.